transforming-growth-factor-beta and Osteoarthritis

Osteoarthritis (OA) is a chronic degenerative joint disorder that leads to disability and affects more than 500 million population worldwide. OA was believed to be caused by the wearing and tearing of articular cartilage, but it is now more commonly referred to as a chronic whole-joint disorder that is initiated with biochemical and cellular alterations in the synovial joint tissues, which leads to the histological and structural changes of the joint and ends up with the whole tissue dysfunction. Currently, there is no cure for OA, partly due to a lack of comprehensive understanding of the pathological mechanism of the initiation and progression of the disease. Therefore, a better understanding of pathological signaling pathways and key molecules involved in OA pathogenesis is crucial for therapeutic target design and drug development. In this review, we first summarize the epidemiology of OA, including its prevalence, incidence and burdens, and OA risk factors. We then focus on the roles and regulation of the pathological signaling pathways, such as Wnt/β-catenin, NF-κB, focal adhesion, HIFs, TGFβ/ΒΜP and FGF signaling pathways, and key regulators AMPK, mTOR, and RUNX2 in the onset and development of OA. In addition, the roles of factors associated with OA, including MMPs, ADAMTS/ADAMs, and PRG4, are discussed in detail. Finally, we provide updates on the current clinical therapies and clinical trials of biological treatments and drugs for OA. Research advances in basic knowledge of articular cartilage biology and OA pathogenesis will have a significant impact and translational value in developing OA therapeutic strategies.

Topics: Cartilage, Articular; Humans; NF-kappa B; Osteoarthritis; Signal Transduction; Transforming Growth Factor beta

Osteoarthritis is a heterogeneous joint disorder that lacks a clinically meaningful disease modifying drug. Animal models have been beneficial in understanding basic joint pathology and providing rationale for future clinical trials on identified targets. This review aims to discuss promising therapeutic targets of osteoarthritis that are currently in animal studies or early clinical trials.. PubMed was searched for articles published between 2017 and 2021 with the following terms: (osteoarthritis AND autophagy) OR (osteoarthritis AND senescence) OR (osteoarthritis AND TGFbeta) OR (osteoarthritis AND EGFR) OR (osteoarthritis AND Wnt/β-catenin) OR (osteoarthritis AND inflammation). Specific targets include the PI3/AKT/mTOR pathway, epidermal growth factor receptor, Toll-like receptors, and inflammatory interleukins, among others.. In reviewing these targets, it is clear that the field of therapeutic targets for osteoarthritis has grown tremendously. We have gained a better understanding of previously identified targets, identified new targets, and have the opportunity to explore enhanced drug delivery via viral vectors. Regardless, translation to clinical benefits is still lacking in most cases. We propose that this may be due to the heterogeneous nature of the disease, lack of early diagnostic markers, mismatched preclinical animal models and clinical populations, and the complex role of many targets of interest.

Topics: Animals; Osteoarthritis; Transforming Growth Factor beta

Osteoarthritis (OA), although extensively researched, still lacks an effective and safe treatment. The only current treatment option available for advanced OA is joint replacement surgery. This surgery may pose the risks of persistent pain, surgical complications and limited implant lifespan. Transforming growth factor (TGF)‑β has a crucial role in multiple cellular processes such as cell proliferation. Any deterioration in TGF‑β signaling pathways can have an immense impact on OA. Owing to the crucial role of TGF‑β in cartilage homeostasis, targeting it could be an alternative therapeutic approach. Additionally, stem cell‑based therapy has recently emerged as an effective treatment strategy that could replace surgery. A number of recent findings suggest that the tissue regeneration effect of stem cells is attributed to the paracrine secretion of anti‑inflammatory and chondroprotective mediators or trophic factors, particularly nanosized extracellular vesicles (i.e., exosomes). Literature searches were performed in the MEDLINE, EMBASE, Cochrane Library and PubMed electronic database for relevant articles published before September 2021. Multiple investigators have confirmed TGF‑β3 as a promising candidate which has the chondrogenic potential to repair articular cartilage degeneration. Combining TGF‑β3 with bone morphogenetic proteins‑6, which has synergistic effect on chondrogenesis, with an efficient platform such as exosomes, which themselves possess a chondroprotective function, offers an innovative and more efficient approach to treat injured cartilage. In addition, multiple findings stating the role of exosomes in chondroprotection has also verified a similar fact showing exosomes may be a more favorable choice than the source itself. In the present review, the importance of TGF‑β family in OA and the possibility of therapeutic treatment using stem cell‑derived exosomes are described.

Topics: Exosomes; Humans; Osteoarthritis; Stem Cells; Transforming Growth Factor beta; Transforming Growth Factors

The effect of the transforming growth factor beta (TGFβ) signaling pathway on joint homeostasis is tissue-specific, non-linear, and context-dependent, representing a unique complexity in targeting TGFβ signaling in joint disease. Here we discuss the variety of mechanisms that TGFβ signaling employs in the synovial joint to maintain healthy joint crosstalk and the ways in which aberrant TGFβ signaling can result in joint degeneration.. Osteoarthritis (OA) epitomizes a condition of disordered joint crosstalk in which multiple joint tissues degenerate leading to overall joint deterioration. Synovial joint tissues, such as subchondral bone, articular cartilage, and synovium, as well as mesenchymal stem cells, each demonstrate aberrant TGFβ signaling during joint disease, whether by excessive or suppressed signaling, imbalance of canonical and non-canonical signaling, a perturbed mechanical microenvironment, or a distorted response to TGFβ signaling during aging. The synovial joint relies upon a sophisticated alliance among each joint tissue to maintain joint homeostasis. The TGFβ signaling pathway is a key regulator of the health of individual joint tissues, and the subsequent interaction among these different joint tissues, also known as joint crosstalk. Dissecting the sophisticated function of TGFβ signaling in the synovial joint is key to therapeutically interrogating the pathway to optimize overall joint health.

Topics: Cartilage, Articular; Humans; Osteoarthritis; Signal Transduction; Synovial Membrane; Transforming Growth Factor beta

The field of osteoarthritis (OA) biology is rapidly evolving and brilliant progress has been made this year as well. Landmark studies of OA biology published in 2021 and early 2022 were selected through PubMed search by personal opinion. These papers were classified by their molecular mechanisms, and it was largely divided into the intracellular signaling mechanisms and the inter-compartment interaction in chondrocyte homeostasis and OA progression. The intracellular signaling mechanisms involving OA progression included (1) Piezo1/transient receptor potential channels of the vanilloid subtype (TRPV) 4-mediated calcium signaling, (2) mechanical load-F-box and WD repeat domain containing 7 (FBXW7) in chondrocyte senescence, (3) mechanical loading-primary cilia-hedgehog signaling, (4) low grade inflammation by toll-like receptor (TLR)-CD14-lipopolysaccharide-binding protein (LBP) complex and inhibitor of NF-κB kinase (IKK) β-nuclear factor kappa B (NF-κB) signaling, (5) selenium pathway and reactive oxygen species (ROS) production, (6) G protein-coupled receptor (GPCR) and cyclic adenosine monophosphate (cAMP) signaling, (7) peroxisome proliferator-activated receptor α (PPARα)-acyl-CoA thioesterase 12 (ACOT12)-mediated de novo lipogenesis and (8) hypoxia-disruptor of telomeric silencing 1-like (DOT1L)-H3-lysine 79 (H3K79) methylation pathway. The studies on inter-compartment or intercellular interaction in OA progression included the following subjects; (1) the anabolic role of lubricin, glycoprotein from superficial zone cells, (2) osteoclast-chondrocyte interaction via exosomal miRNA and sphingosine 1-phosphate (S1P), (3) senescent fibroblast-like synoviocyte and chondrocyte interaction, (4) synovial macrophage and chondrocyte interaction through Flightless I, (5) αV integrin-mediated transforming growth factor beta (TGFβ) activation by mechanical loading, and (6) osteocytic TGFβ in subchondral bone thickening. Despite the disastrous Covid-19 pandemic, many outstanding studies have expanded the boundary of OA biology. They provide both critical insight into the pathophysiology as well as clues for the treatment of OA.

Topics: Biology; Chondrocytes; COVID-19; Hedgehog Proteins; Humans; Ion Channels; NF-kappa B; Osteoarthritis; Pandemics; Thiolester Hydrolases; Transforming Growth Factor beta

Osteoarticular diseases (OD), such as rheumatoid arthritis (RA) and osteoarthritis (OA) are chronic autoimmune/inflammatory and age-related diseases that affect the joints and other organs for which the current therapies are not effective. Cell therapy using mesenchymal stem/stromal cells (MSCs) is an alternative treatment due to their immunomodulatory and tissue differentiation capacity. Several experimental studies in numerous diseases have demonstrated the MSCs' therapeutic effects. However, MSCs have shown heterogeneity, instability of stemness and differentiation capacities, limited homing ability, and various adverse responses such as abnormal differentiation and tumor formation. Recently, acellular therapy based on MSC secreted factors has raised the attention of several studies. It has been shown that molecules embedded in extracellular vesicles (EVs) derived from MSCs, particularly those from the small fraction enriched in exosomes (sEVs), effectively mimic their impact in target cells. The biological effects of sEVs critically depend on their cargo, where sEVs-embedded microRNAs (miRNAs) are particularly relevant due to their crucial role in gene expression regulation. Therefore, in this review, we will focus on the effect of sEVs derived from MSCs and their miRNA cargo on target cells associated with the pathology of RA and OA and their potential therapeutic impact.

Topics: Arthritis, Rheumatoid; Extracellular Vesicles; Humans; Mesenchymal Stem Cell Transplantation; MicroRNAs; Osteoarthritis; Transforming Growth Factor beta

Structural disturbances of the subchondral bone are a hallmark of osteoarthritis (OA), including sclerotic changes, cystic lesions, and osteophyte formation. Osteocytes act as mechanosensory units for the micro-cracks in response to mechanical loading. Once stimulated, osteocytes initiate the reparative process by recruiting bone-resorbing cells and bone-forming cells to maintain bone homeostasis. Osteocyte-expressed sclerostin is known as a negative regulator of bone formation through Wnt signaling and the RANKL pathway. In this review, we will summarize current understandings of osteocytes at the crossroad of allometry and mechanobiology to exploit the relationship between osteocyte morphology and function in the context of joint aging and osteoarthritis. We also aimed to summarize the osteocyte dysfunction and its link with structural and functional disturbances of the osteoarthritic subchondral bone at the molecular level. Compared with normal bones, the osteoarthritic subchondral bone is characterized by a higher bone volume fraction, a larger trabecular bone number in the load-bearing region, and an increase in thickness of pre-existing trabeculae. This may relate to the aberrant expressions of sclerostin, periostin, dentin matrix protein 1, matrix extracellular phosphoglycoprotein, insulin-like growth factor 1, and transforming growth factor-beta, among others. The number of osteocyte lacunae embedded in OA bone is also significantly higher, yet the volume of individual lacuna is relatively smaller, which could suggest abnormal metabolism in association with allometry. The remarkably lower percentage of sclerostin-positive osteocytes, together with clustering of Runx-2 positive pre-osteoblasts, may suggest altered regulation of osteoblast differentiation and osteoblast-osteocyte transformation affected by both signaling molecules and the extracellular matrix. Aberrant osteocyte morphology and function, along with anomalies in molecular signaling mechanisms, might explain in part, if not all, the pre-osteoblast clustering and the uncoupled bone remodeling in OA subchondral bone.

Topics: Animals; Biomarkers; Bone Remodeling; Cartilage, Articular; Disease Susceptibility; Homeostasis; Humans; Joints; Mesenchymal Stem Cells; Osteoarthritis; Osteoblasts; Osteocytes; Signal Transduction; Transforming Growth Factor beta

This meta-analysis was performed to investigate the associations between single-nucleotide polymorphisms (SNPs) in the TGF- β and Smad3 genes and arthritis.. A meta-analysis was performed in STATA 14.0, with publication bias and meta-regression analysis. All types of arthritis were included, and subgroup analyses were performed to interpret variations among different types of arthritis.. Twenty-two qualified studieswere selected to analyze the pooled accuracy, and 4 SNP sites were involved. The analysis of the TGFB1 SNP rs1800470 showed an association with arthritis in allelic (P = 0.011), homozygous (P = 0.034) and recessive (P = 0.021) genetic models. The analysis of the TGFB1 SNP rs1800471 demonstrated a close association with rheumatoid arthritis (RA) in homozygous (P = 0.000, 95%) and recessive (P = 0.008) models. The analysis of the SMAD3 SNP rs12901499 revealed a close association with osteoarthritis (OA) in the allelic (P = 0.001) model.. This research showed that genetic variants of the TGF-β pathway impact arthritis. The polymorphisms rs1800470, rs1800471 and rs12901499 were correlated with a higher prevalence of arthritis.

Topics: Arthritis, Rheumatoid; Gene Frequency; Genetic Association Studies; Genetic Predisposition to Disease; Genotype; Humans; Osteoarthritis; Polymorphism, Single Nucleotide; Smad3 Protein; Transforming Growth Factor beta; Transforming Growth Factor beta1

Osteoarthritis (OA) is a degenerative joint disease characterized by progressive cartilage degradation but also synovial membrane inflammation, osteophyte formation and subchondral bone sclerosis. Medical care is mainly based on alleviating pain symptoms, but to date, no effective drug can stop the disease progression. Cartilage is a tissue composed of only one cell type, chondrocytes, wrapped in a collagen rich extracellular matrix they synthesize. Chondrocytes can adopt different phenotypes in vivo and in vitro, defined by the collagen type they produce. Isolated from their matrix, chondrocytes present the particularity to dedifferentiate, producing fibroblastic type I and III collagens. With OA onset, chondrocytes undergo multiple changes, in terms of proliferation, viability, but also secretory profile. The acquisition of a hypertrophic phenotype (producing aberrant type X collagen and catabolic MMP-13 protease) by chondrocytes is well documented and contributes to OA development. However, it is increasingly believed that chondrocytes rather acquire a variety of degenerated phenotypes at the onset of OA, including a "dedifferentiated-like" phenotype that might also contribute to OA progression. In this review, we will (i) present molecular knowledge underlying dedifferentiation process, (ii) emphasize connections between dedifferentiation and OA and (iii) consider OA therapeutic strategies aiming at the maintenance of chondrogenic phenotype.

Topics: Animals; Cartilage; Cell Dedifferentiation; Chondrocytes; Cytokines; Humans; Osteoarthritis; Signal Transduction; Transforming Growth Factor beta

Osteoarthritis (OA) is the most common joint disease in the United States, affecting more than 30 million people, and is characterized by cartilage degeneration in articulating joints. OA can be viewed as a group of overlapping disorders, which result in functional joint failure. However, the precise cellular and molecular events within which lead to these clinically observable changes are neither well understood nor easily measurable. It is now clear that multiple factors, in multiple joint tissues, contribute to degeneration. Changes in subchondral bone are recognized as a hallmark of OA, but are normally associated with late-stage disease when degeneration is well established. However, early changes such as Bone Marrow Lesions (BMLs) in OA are a relatively recent discovery. BMLs are patterns from magnetic resonance images (MRI) that have been linked with pain and cartilage degeneration. Their potential utility in predicting progression, or as a target for therapy, is not yet fully understood. Here, we will review the current state-of-the-art in this field under three broad headings: (i) BMLs in symptomatic OA: malalignment, joint pain, and disease progression; (ii) biological considerations for bone-cartilage crosstalk in joint disease; and (iii) mechanical factors that may underlie BMLs and drive their communication with other joint tissues. Thus, this review will provide insights on this topic from a clinical, biological, and mechanical perspective. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1818-1825, 2018.

Topics: Animals; Bone Diseases; Bone Marrow; Cartilage Diseases; Cartilage, Articular; Disease Progression; Humans; Knee Joint; Magnetic Resonance Imaging; Osteoarthritis; Osteoarthritis, Knee; Pain; Transforming Growth Factor beta

Osteoarthritis (OA) is one of the most common diseases, affecting more than 10% of populations and thus creating immense socioeconomic burden. The pathological changes of OA involve the entire joint, which is composed of multiple types of tissues and cells, exemplified by cartilage degradation, subchondral bone thickening, osteophyte formation, synovium inflammation and hypertrophy, and ligament degeneration. As joint homeostasis requires a complex network of growth factors to regulate anabolic and catabolic events, the dysregulation of growth factor signalling would have negative impacts on structure and function of multiple joint tissues and eventually lead to the onset and progression of OA. In this review, we will discuss TGF-β, NGF, Hedgehog and Wnt, the four growth factors which have received extensive attention in the field of OA and clinical/translational interrogation about their application in OA therapies.

Topics: Animals; Hedgehog Proteins; Humans; Nerve Growth Factor; Osteoarthritis; Transforming Growth Factor beta; Wnt Signaling Pathway

The objective of this review was to assess the literature for evidence investigating the role of TGF-β in temporomandibular joint disease with osteoarthritis.. An electronic and manual search was carried out on the databases, MEDLINE/PubMed, Cochrane Library, Web Of Science, and EMBASE, from 1975 to December 2015 by two independent evaluators to identify clinical and laboratory trials in English.. The search produced 693 records. Following a process of selection based on certain criteria, eight articles were included.. This systematic review suggests that TGF-β administration alone does not result in joint regeneration; other factors may be involved, such as TGF-β receptor expression ,and TGF-β receptor mutations that do not allow a correct transduction, resulting in TGF-β deficiency. The anabolism induced by this growth factor is also able to neutralize the catabolic processes that are elevated in osteoarthritis. Therefore, further studies are essential to determine how the concentration of TGF-β in the temporomandibular joints acts as a potential marker for the development of degenerative conditions.

Topics: Animals; Biomarkers; Databases, Factual; Humans; Metabolism; Mutation; Osteoarthritis; Receptors, Transforming Growth Factor beta; Temporomandibular Joint; Temporomandibular Joint Disorders; Transforming Growth Factor beta

Musculoskeletal conditions are a major burden on individuals, healthcare systems, and social care systems throughout the world, with indirect costs having a predominant economic impact. Aging is a major contributing factor to the development and progression of arthritic and musculoskeletal diseases. Indeed, aging and inflammation (often referred to as 'inflammaging') are critical risk factors for the development of osteoarthritis (OA), which is one of the most common forms of joint disease. The term 'chondrosenescence' has recently been introduced to define the age-dependent deterioration of chondrocyte function and how it undermines cartilage function in OA. An important component of chondrosenescence is the age-related deregulation of subcellular signaling pathways in chondrocytes. This mini-review discusses the role of age-related alterations in chondrocyte signaling pathways. We focus our attention on two major areas: age-dependent alterations in transforming growth factor-β signaling and changes in protein kinase and phosphoprotein phosphatase activities in aging chondrocytes. A better understanding of the basic signaling mechanisms underlying aging in chondrocytes is likely to facilitate the development of new therapeutic and preventive strategies for OA and a range of other age-related osteoarticular disorders.

Topics: Aged; Aging; Animals; Cartilage, Articular; Cellular Senescence; Chondrocytes; Disease Progression; Humans; Osteoarthritis; Phosphoprotein Phosphatases; Protein Kinases; Signal Transduction; Transforming Growth Factor beta

Despite the tremendous individual suffering and socioeconomic burden caused by osteoarthritis, there are currently no effective disease-modifying treatment options. This is in part because of our incomplete understanding of osteoarthritis disease mechanism. This review summarizes recent developments in therapeutic targets identified from surgical animal models of osteoarthritis that provide novel insight into osteoarthritis pathology and possess potential for progression into preclinical studies.. Several candidate pathways and processes that have been identified include chondrocyte autophagy, growth factor signaling, inflammation, and nociceptive signaling. Major strategies that possess therapeutic potential at the cellular level include inhibiting autophagy suppression and decreasing reactive oxygen species (ROS) production. Cartilage anabolism and prevention of cartilage degradation has been shown to result from growth factor signaling modulation, such as TGF-β, TGF-α, and FGF; however, the results are context-dependent and require further investigation. Pain assessment studies in rodent surgical models have demonstrated potential in employing anti-NGF strategies for minimizing osteoarthritis-associated pain.. Studies of potential therapeutic targets in osteoarthritis using animal surgical models are helping to elucidate osteoarthritis pathology and propel therapeutics development. Further studies should continue to elucidate pathological mechanisms and therapeutic targets in various joint tissues to improve overall joint health.

Topics: Animals; Antirheumatic Agents; Arthritis, Experimental; Autophagy; Cartilage, Articular; Chondrocytes; Humans; Inflammation Mediators; Molecular Targeted Therapy; Osteoarthritis; Signal Transduction; Transforming Growth Factor beta

This review highlights a selection of literature in the area of osteoarthritis biology published between the 2015 and 2016 Osteoarthritis Research Society International (OARSI) World Congress. Highlights were selected from a pubmed search covering cartilage, bone, inflammation and pain. A personal selection was made based, amongst other things, on topics presented during the 2015 conference. This covers circadian rhythm, TGF-β signaling, autophagy, SIRT6, exercise, lubricin, TLR's, pain and NGF. Furthermore, in this review we have made an effort to connect these seemingly distant topics into one scheme of connections between them, revealing a theoretical big picture underneath.

Topics: Animals; Autophagy; Circadian Rhythm; Exercise; Glycoproteins; Humans; Osteoarthritis; Sirtuins; Transforming Growth Factor beta

Transforming growth factor-β (TGFβ) is a pleiotropic cytokine that is important in the regulation of joint homeostasis and disease. TGFβ signalling is induced by loading and has an important function in maintaining the differentiated phenotype of articular chondrocytes. Concentrations of active TGFβ differ greatly between healthy and osteoarthritic joints, being low in healthy joints and high in osteoarthritic joints, leading to the activation of different signalling pathways in joint cells. The characteristic pathology of osteoarthritic joints, such as cartilage damage, osteophyte formation and synovial fibrosis, seems to be stimulated or even caused by the high levels of active TGFβ, in combination with altered chondrocyte signalling pathways (which are also observed in ageing joints). In this Review, the changing role of TGFβ in normal joint homeostasis, ageing and osteoarthritis is discussed: TGFβ counteracts pathological changes in a young healthy joint, alters its signalling during ageing and is a driving force of pathology in osteoarthritic joints.

Topics: Aging; Animals; Humans; Joints; Osteoarthritis; Signal Transduction; Transforming Growth Factor beta

The Wnt signalling pathway is gaining increasing attention in the field of joint pathologies, attributable to its role in the development and homeostasis of the tissues found in the joint, including bone and cartilage. Imbalance in this pathway has been implicated in the development and progression of OA, and interference with the pathway might therefore depict an effective treatment strategy. Though offering multiple opportunities, it is yet to be decided which starting point will bring forth the most promising results. The complexity of the pathway and its interaction with other pathways (such as the TGF-β signalling pathway, which also has a central role in the maintenance of joint homeostasis) means that acting directly on proteins in this signalling cascade entails a high risk of undesired side effects. Therefore, interference with Wnt-induced proteins, such as WISP1, might be an overall more effective and safer therapeutic approach to inhibit the pathological events that take place during OA.

Topics: Cartilage Diseases; CCN Intercellular Signaling Proteins; Cell Communication; Cell Nucleus; Chondrocytes; Cytoplasm; Homeostasis; Humans; Osteoarthritis; Proto-Oncogene Proteins; Receptor Cross-Talk; Transforming Growth Factor beta; Wnt Proteins; Wnt Signaling Pathway

Osteoarthritis (OA) is a common chronic degenerative joint disease, with complicated pathogenic factors and undefined pathogenesis. Various signaling pathways play important roles in OA pathogenesis, including genetic expression, matrix synthesis and degradation, cell proliferation, differentiation, apoptosis, and so on. MicroRNA (miRNA) is a class of non-coding RNA in Eukaryon, regulating genetic expression on the post-transcriptional level. A great number of miRNAs are involved in the development of OA, and are closely associated with different signaling pathways. This article reviews the roles of miRNAs and signaling pathways in OA, looking toward having a better understanding of its pathogenesis mechanisms and providing new therapeutic targets for its treatment.

Topics: Bone Morphogenetic Proteins; Humans; Insulin-Like Growth Factor I; MicroRNAs; NF-kappa B; Osteoarthritis; Signal Transduction; SOX9 Transcription Factor; Transforming Growth Factor beta

Osteoarthritis (OA), the most common form of degenerative joint disease, is linked to high morbidity. It is predicted to be the single greatest cause of disability in the general population by 2030. The development of disease-modifying therapy for OA currently face great obstacle mainly because the onset and development of the disease involve complex molecular mechanisms. In this review, we will comprehensively summarize biological and pathological mechanisms of three key aspects: degeneration of articular cartilage, synovial immunopathogenesis, and changes in subchondral bone. For each tissue, we will focus on the molecular receptors, cytokines, peptidases, related cell, and signal pathways. Agents that specifically block mechanisms involved in synovial inflammation, degeneration of articular cartilage, and subchondral bone remodeling can potentially be exploited to produce targeted therapy for OA. Such new comprehensive agents will benefit affected patients and bring exciting new hope for the treatment of OA.

Topics: Animals; Bone and Bones; Cartilage, Articular; Humans; Osteoarthritis; Signal Transduction; Synovitis; Transforming Growth Factor beta

Osteoarthritis (OA) is a degenerative joint disease that, despite recent progress, has no curative treatment. Considerable research has recently been initiated to identify new potential therapeutic targets. In this review, we will set forth some of the major discoveries in the past 5 years, notably those dealing with the identification of pathogenic factors [hypoxia-inducible factors (HIFs), complement, transforming growth factor (TGF)-β and zinc-ZIP8]. New drugs and concepts currently in clinical development [anti-nerve growth factor (NGF), mesenchymal stromal cells and fibroblast growth factor (FGF)-18] will then be addressed. Finally, we will consider prospective avenues that could lead to mid-to-long-term developments of novel therapeutic concepts, notably those dealing with autophagy regulation and induced pluripotent stem cells.

Topics: Animals; Complement System Proteins; DNA-Binding Proteins; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Osteoarthritis; Transcription Factor MTF-1; Transcription Factors; Transforming Growth Factor beta

To provide an overview of the literature describing the role of asporin, a small leucine-rich proteoglycan (SLRP), in osteoarthritis (OA).. A literature search was performed and reviewed using the narrative approach.. As a class I SLRP member, asporin, is distinct from other SLRPs. Accumulating evidence demonstrates the involvement of asporin in OA pathogenesis. Many human studies have been conducted to explore the association between the D-repeat polymorphisms and OA susceptibility, but these yield inconsistent results. Possible mechanisms for the involvement of asporin in OA pathology include its influence on TGF-β (transforming growth factor-β) signaling pathways and collagen mineralization. To date, no studies were found to use an asporin-deficient animal model that would help to understand disease mechanisms. Many issues must be addressed to clarify the link between asporin and OA to provide a novel therapeutic strategy for OA, perhaps through controlling and modifying the TGF-β-ECM system.. Studies examined demonstrate the involvement of asporin in OA pathogenesis, and possible mechanisms by which asporin may be involved in this process have been proposed. However, large-scale interracial studies should be conducted to investigate the association between asporin and OA, and further investigations are needed to obtain a better understanding of the disease mechanism, develop novel therapeutic strategies, and explore new approaches for diagnosis of OA.

Topics: Extracellular Matrix Proteins; Genetic Predisposition to Disease; Humans; Osteoarthritis; Signal Transduction; Structure-Activity Relationship; Transforming Growth Factor beta

Osteoarthritis (OA) is the most common form of arthritis, resulting in substantial disability and economic burden worldwide. While its exact pathogenesis remains elusive, both in vitro and human population-based studies have merged to support the hypothesis that TGF-β/BMP-mediated signalling pathways play a role in the development of OA. Unraveling the TGF-β/BMP-mediated mechanism(s) in OA has great potential in identifying novel targets and developing new drugs for OA treatment. This review summarizes both in vitro and in vivo evidence of TGF-β/BMP-mediated signal transduction pathways in OA and discusses the future research direction in this regard.

Topics: Bone and Bones; Bone Morphogenetic Proteins; Cartilage, Articular; Chondrocytes; Humans; Osteoarthritis; Osteoblasts; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Synovial fibrosis is often found in OA, contributing heavily to joint pain and joint stiffness, the main symptoms of OA. At this moment the underlying mechanism of OA-related synovial fibrosis is not known and there is no cure available. In this review we discuss factors that have been reported to be involved in synovial fibrosis. The aim of the study was to gain insight into how these factors contribute to the fibrotic process and to determine the best targets for therapy in synovial fibrosis. In this regard, the following factors are discussed: TGF-β, connective tissue growth factor, procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2, tissue inhibitor of metalloproteinase 1, A disintegrin and metalloproteinase domain 12, urotensin-II, prostaglandin F2α and hyaluronan.

Topics: Connective Tissue Growth Factor; Fibrosis; Humans; Joints; Osteoarthritis; Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase; Range of Motion, Articular; Signal Transduction; Synovial Membrane; Transforming Growth Factor beta

During bone resorption, abundant factors previously buried in the bone matrix are released into the bone marrow microenvironment, which results in recruitment and differentiation of bone marrow mesenchymal stem cells (MSCs) for subsequent bone formation, temporally and spatially coupling bone remodeling. Parathyroid hormone (PTH) orchestrates the signaling of many pathways that direct MSC fate. The spatiotemporal release and activation of matrix TGF-β during osteoclast bone resorption recruits MSCs to bone-resorptive sites. Dysregulation of TGF-β alters MSC fate, uncoupling bone remodeling and causing skeletal disorders. Modulation of TGF-β or PTH signaling may reestablish coupled bone remodeling and be a potential therapy.

Topics: Animals; Bone Marrow Cells; Bone Neoplasms; Bone Remodeling; Bone Resorption; Cell Differentiation; Cell Lineage; Gene Expression Regulation; Homeostasis; Humans; Mesenchymal Stem Cells; Osteoarthritis; Osteoclasts; Osteogenesis; Parathyroid Hormone; Signal Transduction; Time Factors; Transforming Growth Factor beta

Osteoarthritis (OA) is the most common degenerative joint disease and no disease-modifying therapy for OA is currently available. Targeting articular cartilage alone may not be sufficient to halt this disease progression. Articular cartilage and subchondral bone act as a functional unit. Increasing evidence indicates that transforming growth factor β (TGFβ) plays a crucial role in maintaining homeostasis of both articular cartilage and subchondral bone. Activation of extracellular matrix (ECM) latent TGFβ at the appropriate time and location is a prerequisite for its function. Aberrant activation of TGFβ in the subchondral bone in response to an abnormal mechanical loading environment induces formation of osteroid islets at the onset of OA. As a result, alteration of subchondral bone structure changes the stress distribution on the articular cartilage and leads to its degeneration. Thus, inhibition of TGFβ activity in the subchondral bone may provide a new avenue of treatment for OA. In this review we will discuss the role of TGFβ in the homeostasis of articular cartilage and subchondral bone as a novel target for OA therapy.

Topics: Animals; Bone and Bones; Bone Remodeling; Cartilage, Articular; Cellular Microenvironment; Homeostasis; Humans; Osteoarthritis; Transforming Growth Factor beta

To give an overview of the literature on the role of small leucine-rich proteoglycans (SLRPs) in osteoarthritis (OA) pathogenesis.. A literature search was performed and reviewed using the narrative approach.. (1) OA is an organ disease with many tissue types and specific roles for each in the pathogenic process. (2) Key biological functions of SLRPs include interacting with collagens to modulate fibril formation, and binding various cell surface receptors and growth factors to influence cellular functions; (3) Accumulating evidence has demonstrated the involvement of SLRPs in OA pathogenesis, most of which came from SLRP-deficient mice models; (4) Possible mechanisms for SLRPs being involved in OA pathogenic process include their roles in the extracellular collagen network, TGF-β signaling pathways, subchondral bone, muscle weakness, and the innate immune inflammation; (5) SLRP-deficient mice offer a potential to understand the molecular mechanisms of OA initiation and progression. (6) Targeting SLRPs may offer a new therapeutic modality for OA through controlling and modifying the TGF-β-ECM system. (7) Monitoring SLRP fragmentation may be a promising biomarker strategy to evaluate OA status.. Recent literature has shown that SLRPs may play an important role in OA pathogenesis. Possible mechanisms by which SLRPs are involved in this process have also been proposed. However, further investigations are needed in this field to better understand its mechanisms, develop treatments to slow down the degenerative process, and explore new approaches for effective and timely diagnosis of OA.

Topics: Animals; Disease Models, Animal; Disease Progression; Humans; Leucine; Mice; Osteoarthritis; Proteoglycans; Signal Transduction; Transforming Growth Factor beta

Osteoarthritis is a progressive joint disease characterized by cartilage degradation and bone remodelling. Under physiologic conditions, articular cartilage displays a stable chondrocyte phenotype, whereas in osteoarthritis a chondrocyte hypertrophy develops near the sites of cartilage surface damage and associates to the pathologic expression of type X collagen. Transglutaminases (TGs) include a family of Ca(2+)-dependent enzymes that catalyze the formation of γ-glutamyl cross-links. Their substrates include a variety of intracellular and extracellular macromolecular components. TGs are ubiquitously and abundantly expressed and implicated in a variety of physiopathological processes. TGs activity is modulated by inflammatory cytokines. TG2 (also known as tissue transglutaminase) mediates the hypertrophic differentiation of joint chondrocytes and interleukin-1-induced calcification. Histomorphometrical and biomolecular investigations document increased TG2 expression in human and experimental osteoarthritis. Consequently, the level of TG2 expression may represent an adjuvant additional marker to monitor tissue remodelling occurring in osteoarthritic joint tissue. Experimental induction of osteoarthritis in TG2 knockout mice is followed from reduced cartilage destruction and increased osteophyte formation compared to wild-type mice, suggesting a different influence on joint bone and cartilage remodelling. The capacity of transamidation by TG2 to regulate activation of latent TGF-β seems to have a potential impact on the regulation of inflammatory response in osteoarthritic tissues. Additional studies are needed to define TG2-regulated pathways that are differently modulated in osteoblasts and chondrocytes during osteoarthritis.

Topics: Animals; Biomarkers; Cartilage, Articular; Femur Head; GTP-Binding Proteins; Humans; Knee Joint; Osteoarthritis; Protein Glutamine gamma Glutamyltransferase 2; Transforming Growth Factor beta; Transglutaminases

Osteoarthritis (OA) is a disease of articular cartilage, with aging as the main risk factor. In OA, changes in chondrocytes lead to the autolytic destruction of cartilage. Transforming growth factor-β has recently been demonstrated to signal not only via activin receptor-like kinase 5 (ALK5)-induced Smad2/3 phosphorylation, but also via ALK1-induced Smad1/5/8 phosphorylation in articular cartilage. In aging cartilage and experimental OA, the ratio ALK1/ALK5 has been found to be increased, and the expression of ALK1 is correlated with matrix metalloproteinase-13 expression. The age-dependent shift towards Smad1/5/8 signalling might trigger the differentiation of articular chondrocytes with an autolytic phenotype.

Topics: Activin Receptors, Type II; Aging; Cell Differentiation; Chondrocytes; Fibrosis; Humans; Osteoarthritis; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad Proteins; Synovial Membrane; Transforming Growth Factor beta

The main purpose of the article is to review recent knowledge about growth factors and their effect on the chondrogenic differentiation of mesenchymal stem cells under in vitro conditions. Damaged or lost articular cartilage leads to progressive debilitation, which have major impact on the life quality of the affected individuals of both sexes in all age groups. Mature hyaline cartilage has a very low self-repair potential due to intrinsic properties - lack of innervation and vascular supply. Another limiting factor is low mitotic potential of chondrocytes. Small defects are healed by migration of chondrocytes, while large ones are healed by formation of inferior fibrocartilage. However, in many cases osteoarthritis develops. Recently, cellular therapy combining mesenchymal stem cells and proper differentiation factors seems to be promising tool for hyaline cartilage defects healing.

Topics: Cell Differentiation; Cell Proliferation; Chondrocytes; Chondrogenesis; Fibroblast Growth Factors; Humans; Hyaline Cartilage; Insulin-Like Growth Factor I; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Osteoarthritis; Transforming Growth Factor beta

Healthy cartilage is maintained by a delicate balance between the anabolic and catabolic activities of articular chondrocytes. This involves actions of numerous cytokines and growth factors that regulate the synthesis and degradation of extracellular matrix components which maintain the functional integrity of the joint. An imbalance between the activities of these anabolic and catabolic factors leads to cartilage degradation resulting in osteoarthritis (OA), a chronic degenerative joint disorder characterized by destruction of articular cartilage, alterations of subchondral bone and synovial fibrosis. Among the cytokines and growth factors that have been studied in the context of cartilage homeostasis and OA, transforming growth factor-beta TGF-beta has emerged as an important molecule that plays a critical role in the development, growth, maintenance and repair of articular cartilage. Deregulation of its signaling and responses has been shown to be involved in OA. Several components of the TGF-beta pathway, including extracellular, cell surface and intracellular molecules, display altered expression or action in OA. In this review, we discuss the regulatory mechanisms of TGF-beta signaling and link these mechanisms to cartilage function, highlighting the important role of TGF-beta in maintaining cartilage function and integrity. We also summarize the alterations in the molecular events of TGF-beta signaling and responses that may contribute to OA progression and discuss the potential of targeting the TGF-beta signaling pathway for the development of novel therapies for OA.

Topics: Animals; Cartilage; Cartilage, Articular; Chondrocytes; Homeostasis; Humans; Joints; Osteoarthritis; Signal Transduction; Transforming Growth Factor beta

Osteoarthritis (OA) constitutes a major health problem. Different signaling pathways are involved that impair homeostasis, but the cross-talk between them (although well investigated and partly understood), remains unclear. HIF-1α promotes chondrocyte differentiation and survival, while HIF-2α coactivates with β-catenin and NF-κB pathways to promote chondrocyte apoptosis and endochondral ossification. Depending on the ALK1/ALK5 ratio in chondrocytes, the TGFβ pathway can play an anabolic or catabolic role. TGFβ1 can activate the β-catenin signaling pathway via ALK5, Smad3, PI3K, and PKA pathways. The mediator Axins balance TGF-β and Wnt/β-catenin signaling during chondrocyte proliferation and maturation. However, the biological functions of Wnt/β-catenin signaling are still controversial. Both excessive and insufficient β-catenin levels may impair the homeostasis of articular chondrocytes by enhancing pathological maturation and apoptosis, respectively; loss- and gain-of-functions of β-catenin cause apoptosis at the center of the joint and chondrocyte maturation at the periphery, depending on the vascularity. The NF-κB transcription factor can be triggered by a host of stress-related stimuli including pro-inflammatory cytokines. The recent discovery of functional cross-regulation between these pathways has shown complex roles for HIF-1α/HIF-2α, TGFβ/BMP, Wnt/β-catenin, and NF-κB signaling pathways in the pathogenesis of OA. This has important implications for potential therapeutic agents directed at these pathways. This review attempts to cover the literature of the past three years dealing with the biology and pathology of the HIF-1α/-2α, TGFβ/BMP, Wnt/β-catenin, and NF-κB/cytokines signaling pathways in OA.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; beta Catenin; Bone Morphogenetic Proteins; Chondrocytes; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; NF-kappa B; Osteoarthritis; Signal Transduction; Transforming Growth Factor beta; Wnt Proteins

Osteoarthritis is characterized by progressive breakdown of articular cartilage. This review summarizes findings of the last year, which shed new light on mechanisms and factors involved in cartilage loss. Evidence is accumulating that the transcription factor hypoxia-inducible factor-2α (HIF-2α) is highly enhanced in OA cartilage and drives catabolic metalloproteinases, including the pivotal MMP-13. In addition, HIF-2α suppresses chondrocyte autophagy, herein promoting chondrocyte apoptosis. The crucial role of MMP-13 is further underlined by reduced OA pathology in MMP-13 deficient mice. An intriguing mechanism to drive MMP-13 production is activation of the chondrocyte discoidin domain receptor (DDR-2) receptor through interaction with denuded collagen type II. The latter might occur in a proteoglycan depleted peri-cellular matrix, where DDR-2 expression is enhanced in OA cartilage and transgenic suppression attenuates experimental OA. The initiating role of ADAMTS-5 in proteoglycan loss appears dependent on interaction with the transmembrane proteoglycan syndecan-4, since syndecan-4 deficient mice are less prone to experimental OA and display reduced ADAMTS-5 activity. Both aging and the osteoarthritis (OA) process itself induce deranged transforming growth factor-β (TGFβ)-receptor expression, causing a shift to dominant usage of the receptor ALK-1, in stead of ALK5 and resulting in a TGFβ mediated catabolic pathway. ALK-1 rather than TGFβ is a promising therapeutic target. Finally, the alarmins S100A8 and 9 have long been considered as markers of inflammatory joint destruction, but now appear to be catabolic mediators.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Calgranulin A; Calgranulin B; Cartilage, Articular; Discoidin Domain Receptors; Humans; Matrix Metalloproteinase 13; Mice; Osteoarthritis; Receptor Protein-Tyrosine Kinases; Receptors, Mitogen; Syndecan-4; Transforming Growth Factor beta

A number of reasonably powered osteoarthritis genome-wide association scans are now in the final phases of their analysis, leaving us all with baited breath. This review highlights some of the osteoarthritis signals and subsequent insights that have emerged from the candidate studies and smaller scale scans that have preceded these more powered studies, and which could therefore be considered as appetizers to the hopeful treats to follow.. If one applies the strict criteria of genome-wide significance thresholds, only two current signals pass muster: GDF5 and 7p22. If one relaxes slightly, other signals emerge, such as DIO2, SMAD3 and ASPN. After these, however, we enter the realm where faith takes precedence.. The search for osteoarthritis susceptibility loci has not been as successful as many had anticipated. This reflects many factors, including the heterogeneous nature of the disease, the tendency to use less severe phenotypes in genetic searches and the reliance on underpowered studies. We do, however, have some successes and in the very near future others will emerge from the more powered scans. Hopefully, combining the current with the new will help our attempts to understand the cause of this complex, common arthritis.

Topics: Animals; Chromosomes, Human, Pair 7; Genetic Predisposition to Disease; Genome-Wide Association Study; Growth Differentiation Factor 5; Humans; Japan; Linkage Disequilibrium; Mice; Netherlands; Osteoarthritis; Polymorphism, Single Nucleotide; Smad3 Protein; Transforming Growth Factor beta; United Kingdom

Transforming growth factor beta (TGFbeta) is a growth factor with many faces. In our osteoarthritis (OA) research we have found that TGFbeta can be protective as well as deleterious for articular cartilage. We postulate that the dual effects of TGFbeta on chondrocytes can be explained by the fact that TGFbeta can signal via different receptors and related Smad signaling routes. On chondrocytes, TGFbeta not only signals via the canonical type I receptor ALK5 but also via the ALK1 receptor. Notably, signaling via ALK5 (Smad2/3 route) results in markedly different chondrocyte responses than ALK1 signaling (Smad1/5/8), and we postulate that the balance between ALK5 and ALK1 expression on chondrocytes will determine the overall effect of TGFbeta on these cells. Importantly, signaling via ALK1, but not ALK5, stimulates MMP-13 expression by chondrocytes. In cartilage of ageing mice and in experimental OA models we have found that the ALK1/ALK5 ratio is significantly increased, favoring TGFbeta signaling via the Smad1/5/8 route, changes in chondrocyte differentiation and MMP-13 expression. Moreover, human OA cartilage showed a significant correlation between ALK1 and MMP-13 expression. In this paper we summarize concepts in OA, its link with ageing and disturbed growth factor responses, and a potential role of TGFbeta signaling in OA development.

Topics: Aging; Animals; Cell Differentiation; Chondrocytes; Humans; Osteoarthritis; Signal Transduction; Transforming Growth Factor beta

Chondrocytes and alteration in chondrocyte differentiation play a central role in osteoarthritis. Chondrocyte differentiation is amongst others regulated by members of the transforming growth factor-beta (TGF-beta) superfamily. The major intracellular signaling routes of this family are via the receptor-Smads. This review is focused on the modulation of receptor-Smad signaling and how this modulation can affect chondrocyte differentiation and potentially osteoarthritis development.. Peer reviewed publications published prior to April 2009 were searched in the Pubmed database. Articles that were relevant for the role of TGF-beta superfamily/Smad signaling in chondrocyte differentiation and for differential modulation of receptor-Smads were selected.. Chondrocyte terminal differentiation is stimulated by Smad1/5/8 activation and inhibited the by Smad2/3 pathway, most likely by modulation of Runx2 function. Several proteins and signaling pathways differentially affect Smad1/5/8 and Smad2/3 signaling. This will result in an altered Smad1/5/8 and Smad2/3 balance and subsequently have an effect on chondrocyte differentiation and osteoarthritis development.. Modulation of receptor-Smads signaling can be expect to play an essential role in both the regulation of chondrocyte differentiation and osteoarthritis development and progression.

Topics: Animals; Cell Differentiation; Cell Proliferation; Chondrocytes; Humans; Mice; Osteoarthritis; Osteogenesis; Signal Transduction; Smad Proteins, Receptor-Regulated; Transforming Growth Factor beta; Ubiquitin-Protein Ligases; Wnt1 Protein

Chondrocyte differentiation and the maintenance of function requires both transient and long-lasting control through humoral factors, particularly under stress, repair and regeneration in vivo or in vitro as in cell and tissue culture. To date, humoral factors from all major classes of molecules are known to contribute: ions (calcium), steroids (estrogens), terpenoids (retinoic acid), peptides (PTHRP, PTH, insulin, FGFs) and complex proteins (IGF-1, BMPs). They may act indirectly through membrane receptors and signal pathways or directly on transcriptional control elements. Those molecules may reach chondrocytes via free diffusion or may be bound to collagens or proteoglycans on extracellular matrix superstructures becoming available on metabolic processing of collagens and/or proteoglycans. Depending on their position in the metabolic cascade controlling chondrocyte development and homeostasis, they may be used in tissue engineering and regenerative approaches towards cartilage repair by direct application, carrier-mediated release or genetic delivery.

Topics: Cartilage, Articular; Cell Differentiation; Chondrocytes; Chondrogenesis; Extracellular Matrix Proteins; Humans; Insulin-Like Growth Factor I; Osteoarthritis; Parathyroid Hormone-Related Protein; Tissue Engineering; Transforming Growth Factor beta; Wound Healing

Once articular cartilage is injured, it has a very limited capacity for self repair. Although current surgical therapeutic procedures for cartilage repair are clinically useful, they cannot restore a normal articular surface. Current research offers a growing number of bioactive reagents, including proteins and nucleic acids, that may be used to augment various aspects of the repair process. As these agents are difficult to administer effectively, gene-transfer approaches are being developed to provide their sustained synthesis at sites of repair. To augment regeneration of articular cartilage, therapeutic genes can be delivered to the synovium or directly to the cartilage lesion. Gene delivery to the cells of the synovial lining is generally considered more suitable for chondroprotective approaches, based on the expression of anti-inflammatory mediators. Gene transfer targeted at cartilage defects can be achieved by either direct vector administration to cells located at or surrounding the defects, or by transplantation of genetically modified chondrogenic cells into the defect. Several studies have shown that exogenous cDNAs encoding growth factors can be delivered locally to sites of cartilage damage, where they are expressed at therapeutically relevant levels. Furthermore, data is beginning to emerge indicating that efficient delivery and expression of these genes is capable of influencing a repair response toward the synthesis of a more hyaline cartilage repair tissue in vivo. This review presents the current status of gene therapy for cartilage healing and highlights some of the remaining challenges.

Topics: Cartilage, Articular; Chondrocytes; Chondrogenesis; Gene Expression; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Humans; Mesenchymal Stem Cell Transplantation; Osteoarthritis; Regeneration; Synovial Membrane; Tissue Engineering; Transforming Growth Factor beta; Wound Healing

Osteophytes are common features of osteoarthritis. This review summarizes the current understanding of the clinical relevance and biology of osteophytes.. This review summarizes peer-reviewed articles published in the PubMed database before May 2006. In addition this review is supplemented with own data and theoretical considerations with regard to osteophyte formation.. Osteophytes can contribute both to the functional properties of affected joints and to clinical relevant symptoms. Osteophyte formation is highly associated with cartilage damage but osteophytes can develop without explicit cartilage damage. Osteophytes are mainly derived from precursor cells in the periosteum and growth factors of the TGFbeta superfamily appear to play a crucial role in their induction.. Osteophyte formation is an integral component of OA pathogenesis and understanding the biology of osteophyte formation can give insights in the disturbed homeostasis in OA joints.

Topics: Animals; Cartilage, Articular; Mice; Osteoarthritis; Periosteum; Transforming Growth Factor beta

While a great deal of information is available on the cellular and molecular biology of cartilage degradation, less is known about anabolism in normal cartilage and degenerating cartilage. A consistent amount of evidence is now available on the neo-synthesis of matrix molecules and enzymes in OA: the entire cell metabolism appears to be increased leading to the hypothesis that chondrocytes in OA are actually "activated". This chapter will focus on anabolic events that are now known to occur in articular cartilage. We will begin to view articular cartilage as a complex three-dimensional tissue in which local events may be different. We will also be interested in viewing the development of degenerative arthritis as a continuum from functionally normal tissue to degeneration.

Topics: Anabolic Agents; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cartilage, Articular; Chondrocytes; Collagen Type II; Humans; Interleukin-1; Osteoarthritis; Transforming Growth Factor beta

Cartilage damage is a major problem in osteoarthritis (OA). Growth factors like transforming growth factor-beta (TGF-beta) have great potential in cartilage repair. In this review, we will focus on the potential therapeutic intervention in OA with TGF-beta, application of the growth factor TGF-beta in cartilage repair and on the side effects of TGF-beta treatment that could occur.. This review summarizes peer-reviewed articles published in the PubMed database before November 2006. In addition, this review is supplemented with recent data of our own group on the use of TGF-beta as a cartilage reparative factor in OA.. TGF-beta is crucial for cartilage maintenance and lack there of results in OA-like changes. Moreover, TGF-beta supplementation can enhance cartilage repair and is therefore a potential therapeutic tool. However, application of TGF-beta supplementation provides problems in other tissues of the joint and results in fibrosis and osteophyte formation. This can potentially be overcome by local inhibition of TGF-beta at sites of unwanted side-effects or by blocking downstream mediators of TGF-beta that are important for the induction of fibrosis or osteophyte formation.. Current understanding of TGF-beta suggests that it essential for cartilage integrity and that it is a powerful tool to prevent or repair cartilage damage. The side-effects that occur with TGF-beta supplementation can be overcome by local inhibition of TGF-beta itself or downstream mediators.

Topics: Animals; Cartilage, Articular; Female; Humans; Male; Mice; Osteoarthritis; Transforming Growth Factor beta

Three years ago we published a book chapter on the role of bone morphogenetic proteins (BMPs) in cartilage repair. Since that time our understanding of the function of osteogenic protein-1 (OP-1) or BMP-7 in cartilage homeostasis and repair has substantially improved and therefore we decided to devote a current review solely to this BMP. Here we summarise the information accumulated on OP-1 from in vitro and ex vivo studies with cartilage cells and tissues as well as from in vivo studies of cartilage repair in various animal models. The primary focus is on articular chondrocytes and cartilage, but data will also be presented on nonarticular cartilage, particularly from the intervertebral disc. The data show that OP-1 is a unique growth factor which, unlike other members of the same BMP family, exhibits in addition to its strong pro-anabolic activity very prominent anti-catabolic properties. Animal studies have demonstrated that OP-1 has the ability to repair cartilage in vivo in various models of articular cartilage degradation, including focal osteochondral and chondral defects and osteoarthritis, as well as models of degeneration in intervertebral disc cartilage. Together our findings indicate a significant promise for OP-1 as therapeutic in cartilage repair.

Topics: Animals; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Cartilage; Disease Models, Animal; Dogs; Homeostasis; Osteoarthritis; Rabbits; Rats; Regeneration; Transforming Growth Factor beta

Difficulties in studying osteoarthritis in humans that stem from both the low sensitivity of diagnostic tools and the low availability of diseased tissues explain why research on animal models remains highly dynamic. This review will summarize the recent advances in this field.. With regard to the etiology of osteoarthritis, synovial macrophages mediate osteophyte formation, whereas increased ligament laxity could be responsible for spontaneous osteoarthritis in guinea pigs. The concomitant changes in subchondral bone and cartilage reported in several models, and the structure-modifying effects of some bone inhibitors have confirmed the importance of bone in osteoarthritis. With regard to cartilage pathobiology, ADAMTS-5 is the major aggrecanase responsible for cartilage destruction, whereas inadequate control of oxidative stress and decreased expression of transforming growth factor-beta receptors could predispose to osteoarthritis. New models include a postmenopausal rat model, the groove model and a joint-specific bone morphogenetic receptor-deficient mouse. The iodoacetate model was also validated as the first pain model of osteoarthritis.. In view of the multiple animal models available, there is a need to reach a consensus on one or several gold standard animal model(s). New studies indicate that important differences in therapeutic response exist between young and old animals, and between spontaneous and surgical models, suggesting that not all models are adequate models of osteoarthritis.

Topics: ADAM Proteins; ADAMTS5 Protein; Animals; Bone and Bones; Cartilage; Disease Models, Animal; Gene Expression Regulation; Guinea Pigs; Humans; Mice; Mice, Transgenic; Osteoarthritis; Oxidative Stress; Pain; Pain Management; Rats; Transforming Growth Factor beta

Osteoarthritis (OA) is the most common form of human arthritis. Genetic factors have been implicated in OA. Through a candidate gene-association study, we found association between asporin and OA. Asporin is an extracellular matrix component expressed abundantly in the articular cartilage of OA patients. A significant association between a polymorphism in the aspartic acid (D) repeat of the asporin gene (ASPN) and knee OA was found; the D14 allele of ASPN is over-represented relative to the common D13 allele, and its frequency increases with disease severity. The D14 allele is also over-represented in patients with hip OA. The association of asporin is replicated in European population by meta-analysis. Asporin suppresses transforming growth factor-beta (TGF-beta)-mediated expression of aggrecan and type II collagen genes and reduced proteoglycan accumulation in an in vitro model of chondrogenesis. Asporin co-localized to and bound to TGF-beta, and inhibited TGF-beta-Smad signal. Clarification of molecular pathway of OA relating to asporin and TGF-beta would lead to order-made medicine and novel therapeutic strategies for OA.

Topics: Alleles; Aspartic Acid; Extracellular Matrix Proteins; Genetic Predisposition to Disease; Humans; Osteoarthritis; Polymorphism, Genetic; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-beta) superfamily members play diverse roles in all aspects of cartilage development and maintenance. It is well established that TGF-betas and bone morphogenetic proteins (BMPs) play distinct roles in the growth plate. This chapter discusses key experiments and experimental approaches that have revealed these roles, and progress toward the identification of previously unsuspected roles. Current understanding of the mechanisms by which different TGF-beta and BMP pathways exert their functions is discussed. Finally attempts to utilize this information to promote cartilage regeneration, and important issues for future research, are outlined.

Topics: Animals; Bone Morphogenetic Proteins; Cell Differentiation; Chondrocytes; Chondrogenesis; Growth Plate; Humans; Mice; Models, Biological; Osteoarthritis; Signal Transduction; Transforming Growth Factor beta

Topics: Animals; Chondrocytes; Chondroitin Sulfates; Humans; Neurons; Osteoarthritis; Transforming Growth Factor beta

Topics: Carrier Proteins; Cell Differentiation; Chondrocytes; Extracellular Matrix Proteins; Genetic Predisposition to Disease; Humans; Multifactorial Inheritance; Osteoarthritis; Transforming Growth Factor beta

In menopausal women and the elderly, populations most often affected by osteoarthritis (OA), estrogen levels are lower than normal, which suggests that estrogen may be an important regulator of OA. Estrogen can influence chondrocyte function on multiple levels by interacting with cellular growth factors, adhesion molecules, and cytokines. Nevertheless, findings regarding a correlation between estrogen and OA are inconsistent and inconclusive and range from estrogen protecting against OA to cartilage damage mediated by high levels of estrogen and higher binding to estrogen receptors. In this review, we summarize current in vivo and in vitro research and discuss future directions for analyses of the role of estrogen in OA.

Topics: Cell Adhesion Molecules; Chondrocytes; Estrogen Replacement Therapy; Estrogens; Female; Humans; Insulin-Like Growth Factor I; Intercellular Adhesion Molecule-1; Osteoarthritis; Receptors, Estrogen; Transforming Growth Factor beta

In osteoarthritic cartilage, chondrocytes are able to present heterogeneous cellular reactions with expression and synthesis of the (pro)collagen types characteristic of prechondrocytes (type IIA), hypertrophic chondrocytes (type X), as well as differentiated (types IIB, IX, XI, VI) and dedifferentiated (types I, III) chondrocytes. The expression of type IIA procollagen in human osteoarthritic cartilage support the assumption that OA chondrocytes reverse their phenotype towards a chondroprogenitor phenotype. Recently, we have shown that dedifferentiation of mouse chondrocytes induced by subculture was associated with the alternative splicing of type II procollagen pre-mRNA with a switch from the IIB to the IIA form. In this context, we demonstrated that BMP-2 favours expression of type IIB whereas TGF-beta1 potentiates expression of type IIA induced by subculture. These data reveal the specific capability of BMP-2 to reverse the program of chondrocyte dedifferentiation. This interesting feature needs to be tested with human chondrocytes since cell amplification is required for the currently used autologous chondrocyte transplantation.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cartilage, Articular; Cell Differentiation; Cells, Cultured; Chondrocytes; Collagen; Extracellular Matrix; Humans; Osteoarthritis; Procollagen; Transforming Growth Factor beta

Growth factors are small proteins that regulate cellular function. Some growth factors, including insulin like growth factor-1 and transforming growth factor-beta play an anabolic role in articular cartilage. The loss of articular cartilage that occurs in osteoarthritis may reflect, in part, an insufficiency of such factors. Recent evidence suggests that inhibition of these growth factors may contribute to the disease process in osteoarthritis. Inhibitors identified to date include binding proteins, cytokines, mechanical factors, and nutritional status. The mechanisms underlying the actions and interactions of these regulatory factors are beginning to be elucidated and may have implications for the development of new approaches to the treatment of osteoarthritis.

Topics: Age Factors; Animals; Biomechanical Phenomena; Cartilage, Articular; Cytokines; Growth Inhibitors; Growth Substances; Humans; Insulin-Like Growth Factor Binding Proteins; Osteoarthritis; Peptide Hydrolases; Somatomedins; Transforming Growth Factor beta

Matrix metalloproteinase (MMP) plays an important role in degradation of cartilage matrix. The expression of MMPs in cartilage or syovial membrane was increased in osteoarthritis or rheumatoid arthritis. We summarized the regulation mechanism of MMP production, and described pharmacologic inhibitors such as non steroidal anti-inflammatory drugs, steroid and growth factors, which might be useful to prevention of joint destruction.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Arthritis, Rheumatoid; Cartilage, Articular; Depression, Chemical; Dexamethasone; Extracellular Matrix; Humans; Insulin-Like Growth Factor I; Matrix Metalloproteinases; Osteoarthritis; Synovial Membrane; Transforming Growth Factor beta

Inefficient healing of bony and cartilaginous defects is a common situation encountered by orthopedic surgeons. Enhancing the regenerative potential of bone and articular cartilage has the potential for profound applications in treatment of nonunions, large segmental bone and cartilage defects, and arthritis. The bone morphogenetic proteins (BMPs) encode a highly conserved class of signaling factors that possess the ability to induce ectopic cartilage and bone formation in vivo. Bone morphogenetic protein family members are expressed during limb development, endochondral ossification, and early fracture and cartilage repair. Loss-of-function and gain-of-function studies have demonstrated the necessity and sufficiency of these genes, respectively, in regulating both cartilage and bone development. Several recent animal studies have demonstrated the potential of BMPs to enhance spinal fusion, repair critical-size defects, accelerate union, and heal articular cartilage lesions. A limited number of clinical trials using BMPs in human beings have been reported, and these agents are currently available for clinical use within and outside the United States. Current challenges to be met are the development of efficient delivery systems to present BMP proteins or genes to target sites and to enhance their duration and function at these locations.

Topics: Animals; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Cartilage, Articular; Cells, Cultured; Drug Delivery Systems; Fractures, Bone; Humans; Osteoarthritis; Spinal Fusion; Transforming Growth Factor beta; Wound Healing

Topics: Adult; Animals; Arthritis, Rheumatoid; Bone Marrow Transplantation; Bone Morphogenetic Proteins; Bone Regeneration; Cartilage, Articular; Chondrocytes; Cytokines; Down-Regulation; Gene Transfer Techniques; Humans; Mice; Mice, SCID; Middle Aged; Osteoarthritis; Proteoglycans; Rabbits; Rats; Rats, Inbred Lew; Rats, Nude; Recombinant Proteins; Regeneration; Stem Cell Transplantation; Stem Cells; Transforming Growth Factor beta

Osteoarthritis is a joint disease that is characterized by focal degradation of articular cartilage. In addition to the degeneration of articular cartilage, attempts at repair are found in the affected tissue. Cartilage cells (chondrocytes) play a key role, not only in the destructive process, but also in the repair response. It has become apparent that anabolic and catabolic mediators, released from chondrocytes themselves or from other joint cells, drive both destructive and repair activities in the osteoarthritic joint.

Topics: Bone Morphogenetic Proteins; Cartilage, Articular; Growth Differentiation Factor 5; Growth Substances; Humans; Insulin-Like Growth Factor I; Interleukin-1; Nitric Oxide; Osteoarthritis; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Activated articular chondrocytes produce large amounts of nitric oxide (NO), and there is increasing evidence that this is involved in the etiopathogenesis of osteoarthritis (OA). Because of its short half-life, the biological effects of endogenously produced NO are likely to occur locally within the cartilage. We have observed that inhibitors of NO synthases relieve the inhibition of matrix synthesis that otherwise occurs in response to IL-1. To avoid the use of inhibitors, we have recently transduced chondrocytes with the iNOS (NOS-2) gene and confirmed the ability of the endogenously produced NO to inhibit matrix synthesis. Despite the high levels of NO made by these cells, there was no evidence of apoptosis or other forms of cell death. NO was also shown to inhibit the production of TGF-beta(1)by cells treated with IL-1, as well as to decrease matrix production in response to IGF-1. The hypothesis that NO inhibits matrix production by interfering with important autocrine and paracrine factors should be entertained.

Topics: Chondrocytes; Humans; Nitric Oxide; Osteoarthritis; Proteoglycans; Transforming Growth Factor beta

Cartilage destruction in arthritis and osteoarthritis is linked to aberrant cytokine and growth factor expression in the affected tissues. It becomes clear that the balance of protective and destructive cytokines is more important for the net destruction than the absolute levels of destructive mediators. IL-1 is a key destructive mediator in arthritis and probably also in osteoarthritis. Production of the cartilage destructive enzyme stromelysin is linked to IL-1. In osteoarthritis, excessive formation of the growth factor TGF beta may contribute to cartilage lesions and osteophyte formation, in particular. Therapy should be aimed at neutralization of IL-1 and stimulation of safe anabolic growth factors for the articular cartilage, such as IGF-1 and the novel bone and cartilage derived morphogenetic proteins.

Topics: Animals; Arthritis, Rheumatoid; Cytokines; Growth Inhibitors; Growth Substances; Humans; Interleukin-1; Osteoarthritis; Transforming Growth Factor beta

Topics: Animals; Cartilage, Articular; Humans; Osteoarthritis; Rabbits; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

Growth-hormones like Insulin-Like-Growth-Hormone-1 (IGF-1) and Tissue-Growth-Factor-beta (TGF-beta) and Cytokines IL-1, IL-6 and TGF-alpha play an important part in the homeostasis and also degradation of the articular cartilage. IL-1 stimulates the production of proteolytic enzymes and inhibits the synthesis of aggrecan and leads therefore to a degradation of the cartilage. TNF-alpha acts in a similar way, whereas the significance of IL-6 for chondrocytes is not yet fully understood. TGF-beta and IGF-1 in contrast have a positive influence on cartilage metabolism, f. ex. TGF-beta stimulates the synthesis of the natural occurring metalloproteinase-inhibitor (TIMP).

Topics: Cartilage, Articular; Cytokines; Glycoproteins; Homeostasis; Humans; Insulin-Like Growth Factor I; Interleukin-1; Interleukin-6; Osteoarthritis; Tissue Inhibitor of Metalloproteinases; Transforming Growth Factor alpha; Transforming Growth Factor beta

Polypeptide growth factors play a major role in the regulation of cell behavior, including that of articular chondrocytes. Among the most influential of these factors identified for articular cartilage are insulin like growth factor I (IGF-I), basic fibroblast growth factor (bFGF), and transforming growth factor beta (TGF beta). IGF-I stimulates articular chondrocyte matrix synthetic and mitotic activity and inhibits chondrocyte mediated matrix catabolism. The role of bFGF as a potent mitogen for articular chondrocytes is well established. In contrast, this factor appears to play a complex role in matrix synthesis and degradation, promoting both anabolic and catabolic functions. Both IGF-I and bFGF have been shown to augment articular cartilage repair in vivo. TGF beta is particularly dependent upon the context in which it acts, eliciting seemingly opposite effects under different experimental conditions. These and other factors interact to modulate their respective actions, creating effector cascades and feedback loops of intercellular and intracellular events that control articular chondrocyte functions. Elucidation of the actions and interactions of these factors may be expected to clarify the etiopathogenesis of osteoarthritis and possibly offer novel methods for its treatment.

Topics: Cartilage, Articular; Fibroblast Growth Factor 2; Growth Substances; Humans; Insulin-Like Growth Factor I; Osteoarthritis; Transforming Growth Factor beta

Human bone contains an abundance of polypeptide growth factors. These growth factors stimulate the proliferation and activity of bone cells and can stimulate bone formation. Data from this laboratory and others suggest that bone growth factors may act to couple bone formation to resorption to maintain bone mass during remodeling. Research is underway to study these growth factors in bones and teeth, and their possible roles in both the pathogenesis and the treatments of osteoporosis and dental diseases.

Topics: Animals; Bone and Bones; Bone Remodeling; Dentin; Estrogens; Growth Disorders; Growth Substances; Humans; Odontoblasts; Odontogenesis; Osteoarthritis; Osteogenesis; Osteoporosis; Periodontal Diseases; Somatomedins; Tooth; Transforming Growth Factor beta; Vitamin D Deficiency

To investigate the therapeutic effect of oral glucosamine (GS) as an adjunct to hyaluronic acid (HA) injection on patients with temporomandibular joint osteoarthritis (TMJ OA).. In this clinical trial, 136 participants, diagnosed as TMJ OA clinically and radiographically, were enrolled and randomized into two groups (group GS + HA: oral GS + HA injection; group placebo + HA: oral placebo + HA injection). Pain, maximum interincisal mouth opening (MMO), the levels of IL-1β, IL-6, and TGF-β in TMJ synovial were defined as the outcome measurements and conducted before operation, and at 1-month and 1-year follow-up.. In both groups, pain scores were decreased and MMOs were increased at 1-month and 1-year follow-up, the changes at 1-year follow-up showed statistically significant intergroup differences. At 1-month follow-up, only IL-6 concentration was lower in group GS + HA than that in group placebo + HA. One year later, TGF-β concentration was higher and IL-6 and IL-1β concentrations were lower in group GS + HA than those in group placebo + HA.. Both strategies alleviated symptoms in short term, but the patients treated with GS benefited more than those with placebo in long term, which may be due to the suppression of IL-1β and IL-6 and the stimulation of TGF-β.

Topics: Administration, Oral; Adult; Dietary Supplements; Double-Blind Method; Drug Therapy, Combination; Female; Glucosamine; Humans; Hyaluronic Acid; Injections, Intra-Articular; Interleukin-1beta; Interleukin-6; Male; Middle Aged; Musculoskeletal Pain; Osteoarthritis; Pain Measurement; Prospective Studies; Synovial Fluid; Temporomandibular Joint; Transforming Growth Factor beta; Viscosupplements; Young Adult

Osteoarthritis is a long-term complication of acute articular infections. However, the roles of cartilage and synovia in this process are not yet fully understood.. Patients with acute joint infections were enrolled in a prospective clinical trial and the cytokine composition of effusions compared in patients with arthroplasty (n = 8) or with intact joints (n = 67). Cytokines and cell function were also analyzed using a human in vitro model of joint infection.. Synovial IL-1β levels were significantly higher in patients with arthroplasty (p = 0.004). Higher IL-1β concentrations were also found in the in vitro model without chondrocytes (p < 0.05). The anti-inflammatory cytokines IL-4 and IL-10 were consistently expressed in vivo and in vitro, showing no association with the presence of cartilage or chondrocytes. In contrast, FasL levels increased steadily in vitro, reaching higher levels without chondrocytes (p < 0.05). Likewise, the viability of synovial fibroblasts (SFB) during infection was higher in the presence of chondrocytes. The cartilage-metabolism markers aggrecan and bFGF were at higher concentrations in intact joints, but also synthesized by SFB.. Our data suggest an anti-inflammatory effect of cartilage associated with the SFBs' increased resistance to infections, which displayed the ability to effectively synthesize cartilage metabolites.The trial is registered with DRKS 00003536, MISSinG.

Topics: Acute Disease; Aged; Aggrecans; Cartilage; Chondrocytes; Female; Fibroblast Growth Factor 2; Humans; Interleukin-1beta; Male; Middle Aged; Osteoarthritis; Prospective Studies; Synovial Fluid; Transforming Growth Factor beta

Osteoarthritis (OA) is a serious consequence of focal osteochondral defects. Gene transfer of human transforming growth factor beta (hTGF-β) with recombinant adeno-associated virus (rAAV) vectors offers a strategy to improve osteochondral repair. However, the long-term in vivo effects of such rAAV-mediated TGF-β overexpression including its potential benefits on OA development remain unknown.. Focal osteochondral defects in minipig knees received rAAV-lacZ (control) or rAAV-hTGF-β in vivo. After one year, osteochondral repair and perifocal OA were visualized using validated macroscopic scoring, ultra-high-field MRI at 9.4 T, and micro-CT. A quantitative estimation of the cellular densities and a validated semi-quantitative scoring of histological and immunohistological parameters completed the analysis of microarchitectural parameters.. Direct rAAV-hTGF-β application induced and maintained significantly improved defect filling and safranin O staining intensity and overall cartilage repair at one year in vivo. In addition, rAAV-hTGF-β led to significantly higher chondrocyte densities within the cartilaginous repair tissue without affecting chondrocyte hypertrophy and minimized subarticular trabecular separation. Of note, rAAV-hTGF-β significantly improved the adjacent cartilage structure and chondrocyte density and reduced overall perifocal OA development after one year in vivo.. rAAV-hTGF-β treatment improves long-term osteochondral repair and delays the progression of perifocal OA in a translational model. These findings have considerable potential for targeted molecular approaches to treat focal osteochondral defects.

Topics: Animals; Cartilage, Articular; Dependovirus; Humans; Models, Animal; Osteoarthritis; Swine; Swine, Miniature; Transforming Growth Factor beta

TGF-β signaling is crucial for modulating osteoarthritis (OA), and protein phosphatase magnesium-dependent 1A (PPM1A) has been reported as a phosphatase of SMAD2 and regulates TGF-β signaling, while the role of PPM1A in cartilage homeostasis and OA development remains largely unexplored. In this study, we found increased PPM1A expression in OA chondrocytes and confirmed the interaction between PPM1A and phospho-SMAD2 (p-SMAD2). Importantly, our data show that PPM1A KO substantially protected mice treated with destabilization of medial meniscus (DMM) surgery against cartilage degeneration and subchondral sclerosis. Additionally, PPM1A ablation reduced the cartilage catabolism and cell apoptosis after the DMM operation. Moreover, p-SMAD2 expression in chondrocytes from KO mice was higher than that in WT controls with DMM induction. However, intraarticular injection with SD-208, repressing TGF-β/SMAD2 signaling, dramatically abolished protective phenotypes in PPM1A-KO mice. Finally, a specific pharmacologic PPM1A inhibitor, Sanguinarine chloride (SC) or BC-21, was able to ameliorate OA severity in C57BL/6J mice. In summary, our study identified PPM1A as a pivotal regulator of cartilage homeostasis and demonstrated that PPM1A inhibition attenuates OA progression via regulating TGF-β/SMAD2 signaling in chondrocytes and provided PPM1A as a potential target for OA treatment.

Topics: Animals; Chondrocytes; Mice; Mice, Inbred C57BL; Osteoarthritis; Phosphoprotein Phosphatases; Protein Phosphatase 2C; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta

Patients with temporomandibular dysfunction (TMD) usually suffer from pathology or malpositioning of the temporomandibular joint (TMJ) disk, leading to the degenerative lesion of condyles. Kartogenin can promote the repair of damaged cartilage. This study aimed to explore whether intra-articular injection of kartogenin could alleviate the TMJ injury induced by type II collagenase. We measured the head withdrawal threshold and found that kartogenin alleviated the pain around TMD induced by type II collagenase. We observed the morphology of the condylar surface and found that kartogenin protected the integration of the condylar surface. We analyzed the density of the subchondral bone and found that kartogenin minimized the damage of TMJ injury to the subchondral bone. We next explored the histological changes and found that kartogenin increased the thickness of the proliferative layer and more collagen formation in the superficial layer. Then, to further ensure whether kartogenin promotes cell proliferation in the condyle, we performed immunohistochemistry of proliferating cell nuclear antigen (PCNA). The ratio of PCNA-positive cells was significantly increased in the kartogenin group. Next, immunofluorescence of TGF-β1 and SMAD3 was performed to reveal that kartogenin activated the TGF-β/SMAD pathway in the proliferative layer. In conclusion, kartogenin may have a therapeutic effect on TMJ injury by promoting cell proliferation in cartilage and subchondral bone. Kartogenin may be promising as an intra-articular injection agent to treat TMD.

Topics: Animals; Cartilage, Articular; Chondrocytes; Collagenases; Humans; Mandibular Condyle; Osteoarthritis; Proliferating Cell Nuclear Antigen; Rats; Temporomandibular Joint; Temporomandibular Joint Disorders; Transforming Growth Factor beta

Basic Calcium Phosphate (BCP) crystals play an active role in the progression of osteoarthritis (OA). However, the cellular consequences remain largely unknown. Therefore, we characterized for the first time the changes in the protein secretome of human OA articular chondrocytes as a result of BCP stimulation using two unbiased proteomic analysis methods.. Isolated human OA articular chondrocytes were stimulated with BCP crystals and examined by Quantitative Reverse Transcription PCR (RT-qPCR) and enzyme-linked immune sorbent assay (ELISA) after twenty-four and forty-eight hours. Forty-eight hours conditioned media were analyzed by label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) and an antibody array. The activity of BCP dependent Transforming Growth Factor Beta (TGF-β) signaling was analyzed by RT-qPCR and luciferase reporter assays. The molecular consequences regarding BCP-dependent TGF-β signaling on BCP-dependent Interleukin 6 (IL-6) were investigated using specific pathway inhibitors.. Synthesized BCP crystals induced IL-6 expression and secretion upon stimulation of human articular chondrocytes. Concomitant induction of catabolic gene expression was observed. Analysis of conditioned media revealed a complex and diverse response with a large number of proteins involved in TGF-β signaling, both in activation of latent TGF-β and TGF-β superfamily members, which were increased compared to non-stimulated OA chondrocytes. Activity of this BCP driven TGF-β signaling was confirmed by increased activity of expression of TGF-β target genes and luciferase reporters. Inhibition of BCP driven TGF-β signaling resulted in decreased IL-6 expression and secretion with a moderate effect on catabolic gene expression.. BCP crystal stimulation resulted in a complex and diverse chondrocyte protein secretome response. An important role for BCP-dependent TGF-β signaling was identified in development of a pro-inflammatory environment.

Topics: Calcium Phosphates; Chondrocytes; Chromatography, Liquid; Culture Media, Conditioned; Humans; Interleukin-6; Osteoarthritis; Proteomics; Secretome; Signal Transduction; Tandem Mass Spectrometry; Transforming Growth Factor beta

Synovial fibrosis is a driver in the progression of osteoarthritis (OA). Fibroblast growth factor 10 (FGF10) has prominent anti-fibrotic effects in many diseases. Thus, we explored the anti-fibrosis effects of FGF10 in OA synovial tissue. In vitro, fibroblast-like synoviocytes (FLSs) were isolated from OA synovial tissue and stimulated with TGF-β to establish a cell model of fibrosis. After treatment with FGF10, we assessed the effects on FLS proliferation and migration using CCK-8, EdU, and scratch assays, and collagen production was observed using Sirius Red Stain. The JAK2/STAT3 pathway and expression of fibrotic markers were evaluated through western blotting (WB) and immunofluorescence (IF). In vivo, we treated mice with OA induced by surgical destabilization of the medial meniscus (DMM) with FGF10 and assessed the anti-OA effect using histological and immunohistochemical (IHC) staining of MMP13, and fibrosis was evaluated using HE and Masson's trichrome staining. The expression of IL-6/JAK2/STAT3 pathway components was determined using ELISA, WB, IHC, and IF. In vitro, FGF10 inhibited TGF-β-induced FLS proliferation and migration, decreased collagen deposition, and improved synovial fibrosis. Moreover, FGF10 mitigated synovial fibrosis and improved the symptoms of OA in DMM-induced OA mice. Overall, FGF10 had promising anti-fibrotic effects on FLSs and improved OA symptoms in mice. The IL-6/STAT3/JAK2 pathway plays key roles in the anti-fibrosis effect of FGF10. This study is the first to demonstrate that FGF10 inhibited synovial fibrosis and attenuated the progression of OA by inhibiting the IL-6/JAK2/STAT3 pathway.

Topics: Animals; Fibroblast Growth Factor 10; Fibroblasts; Interleukin-6; Mice; Osteoarthritis; Synovial Membrane; Transforming Growth Factor beta

Molecular transport between the circulatory and musculoskeletal systems regulates articular joint physiology in health and disease. Osteoarthritis (OA) is a degenerative joint disease linked to systemic and local inflammation. Inflammatory events involve cytokines, which are secreted by cells of the immune system and modulate molecular transport across tissue interfaces (referred to as tight junction [TJ] barrier function). In a previous study from our group, OA knee joint tissues were shown to exhibit size separation of different sized molecules delivered as a single bolus to the heart (Ngo et al. in Sci. Rep. 8:10254, 2018). Here, in a follow up study of parallel design, we test the hypothesis that two common cytokines, with multifaceted roles in the etiology of osteoarthritis as well as immune state in general, modulate the barrier function properties of joint tissue interfaces. Specifically, we probe the effect of an acute cytokine increase (spike) on molecular transport within tissues and across tissue interfaces of the circulatory and musculoskeletal systems. A single bolus of fluorescent-tagged 70 kDa dextran, was delivered intracardially, either alone, or with either the pro-inflammatory cytokine TNF-α or the anti-inflammatory cytokine TGF-β, to skeletally mature (11 to 13-month-old) guinea pigs (Dunkin-Hartley, a spontaneous OA animal model). After five minutes' circulation, whole knee joints were serial sectioned and fluorescent block face cryo-imaged at near-single-cell resolution. The 70 kDa fluorescent-tagged tracer is analogous in size to albumin, the most prevalent blood transporter protein, and quantification of tracer fluorescence intensity gave a measure of tracer concentration. Within five minutes, a spike (acute doubling) in circulating cytokines TNF-α or TGF-β significantly disrupted barrier function between the circulatory and musculoskeletal systems, with barrier function essentially abrogated in the TNF-α group. In the entire volume of the joint (including all tissue compartments and the bounding musculature), tracer concentration was significantly decreased in the TGF-β- and TNF-α- compared to the control-group. These studies implicate inflammatory cytokines as gatekeepers for molecular passage within and between tissue compartments of our joints and may open new means to delay the onset and mitigate the progression of degenerative joint diseases such as OA, using pharmaceutical and/or physical measures.

Topics: Animals; Cytokines; Follow-Up Studies; Guinea Pigs; Osteoarthritis; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Transforming growth factor-β (TGF-β) signaling via SMAD2/3 is crucial to control cartilage homeostasis. However, TGF-β can also have detrimental effects by signaling via SMAD1/5/9 and thereby contribute to diseases like osteoarthritis (OA). In this study, we aimed to block TGF-β-induced SMAD1/5/9 signaling in primary human OA chondrocytes, while maintaining functional SMAD2/3 signaling.. Human OA chondrocytes were pre-incubated with different concentrations of ALK4/5/7 kinase inhibitor SB-505124 before stimulation with TGF-β. Changes in SMAD C-terminal phosphorylation were analyzed using Western blot and response genes were measured with quantitative Polymerase Chain Reaction. To further explore the consequences of our ability to separate pathways, we investigated TGF-β-induced chondrocyte hypertrophy.. Pre-incubation with 0.5 µM SB-505124, maintained ±50% of C-terminal SMAD2/3 phosphorylation and induction of JUNB and SERPINE1, but blocked SMAD1/5/9-C phosphorylation and expression of ID1 and ID3. Furthermore, TGF-β, in levels comparable to those in the synovial fluid of OA patients, resulted in regulation of hypertrophic and dedifferentiation markers in OA chondrocytes; i.e. an increase in COL10, RUNX2, COL1A1, and VEGF and a decrease in ACAN expression. Interestingly, in a subgroup of OA chondrocyte donors, blocking only SMAD1/5/9 caused stronger inhibition on TGF-β-induced RUNX2 than blocking both SMAD pathways.. Our findings indicate that using low dose of SB-505124 we maintained functional SMAD2/3 signaling that blocks RUNX2 expression in a subgroup of OA patients. We are the first to show that SMAD2/3 and SMAD1/5/9 pathways can be separately modulated using low and high doses of SB-505124 and thereby split TGF-β's detrimental from protective function in chondrocytes.

Topics: Cartilage, Articular; Chondrocytes; Core Binding Factor Alpha 1 Subunit; Humans; Osteoarthritis; Phosphorylation; Smad2 Protein; Transforming Growth Factor beta

We aimed to explore the effect and mechanism of the Src inhibitor PP2 on osteoarthritis (OA) progression.. The protein expressions of Src, p-Src (y418) and p-FAK in normal and OA human chondrocytes were detected by immunofluorescence (IF) analysis. Chondrocytes from the femur and tibial plateau of 3-day-old mice were extracted and inoculated into 6-well plates. The chondrocytes were co-cultured with IL-1β and different doses of PP2, and then the degeneration of extracellular matrix was analyzed. A mouse OA model was induced by destabilizing medial meniscectomy of the right knee. Two weeks after the operation, different doses of PP2 were injected intraperitoneally. The drug was given three times a week for 6 weeks, and then the mice were sacrificed. Histopathological, IF and immunoblotting analyses were used to detect key OA catabolic markers and protein expression and related signaling.. The levels of Src, p-Src (y418) and p-FAK in the knee cartilage tissue of patients with OA were abnormally increased. After chondrocytes were co-treated with IL-1β and different doses of PP2, the results showed that PP2 reduced the abnormal increase of β-catenin, p-β-catenin and other proteins induced by IL-1β, and reversed the decrease of p-Smad3, aggrecan and collagen Ⅱ protein levels. Meanwhile, intraperitoneal injection of PP2 in vivo significantly reduced the degeneration of articular cartilage in the OA mouse model.. Our data indicate that targeting Src with PP2 protected against cartilage destruction in an OA mouse model by inhibiting Wnt/β-catenin and activating TGF-β/Smad signaling, suggesting that Src may be a potential therapeutic target for OA treatment.

Topics: Animals; beta Catenin; Cartilage, Articular; Cells, Cultured; Chondrocytes; Disease Models, Animal; Humans; Mice; Osteoarthritis; Transforming Growth Factor beta; Wnt Signaling Pathway

Osteoarthritis (OA) is a degenerative joint disease associated with excessive mechanical loading. The aim here was to elucidate whether different subpopulations of chondrocytes exhibit distinct phenotypes in response to variations in loading conditions. Furthermore, we seek to investigate the transcriptional switches and cell crosstalk among these chondrocytes subsets.. Proteomic analysis was performed on cartilage tissues isolated from weight-bearing and non-weight-bearing regions. Additionally, single-cell RNA sequencing was employed to identify different subsets of chondrocytes. For disease-specific cells, in vitro differentiation induction was performed, and their presence was confirmed in human cartilage tissue sections using immunofluorescence. The molecular mechanisms underlying transcriptional changes in these cells were analysed through whole-transcriptome sequencing.. In the weight-bearing regions of OA cartilage tissue, a subpopulation of chondrocytes called OA hypertrophic chondrocytes (OAHCs) expressing the marker genes SLC39A14 and COL10A1 are present. These cells exhibit unique characteristics of active cellular interactions mediated by the TGFβ signalling pathway and express OA phenotypes, distinct from hypertrophic chondrocytes in healthy cartilage. OAHCs are mainly distributed in the superficial region of damaged cartilage in human OA tissue, and on TGFβ stimulation, exhibit activation of transcriptional expression of iron metabolism-related genes, along with enrichment of associated pathways.. This study identified and validated the existence of a subset of OAHCs in the weight-bearing area of OA cartilage tissue. Our findings provide a theoretical basis for targeting OAHCs to slow down the progression of OA and facilitate the repair of cartilage injuries.

Topics: Cartilage, Articular; Chondrocytes; Humans; Osteoarthritis; Phenotype; Proteomics; Transforming Growth Factor beta

In osteoarthritis (OA), synovial pathology may be induced by proteins released from degenerated cartilage. This study was conducted to identify the proteins released from OA cartilage. OA cartilage was obtained from OA knees at macroscopically preserved areas (PRES) and degenerated areas (DEG), while control cartilage (CONT) was collected from non-arthritic knees. Released proteins were obtained from these cartilage samples by repeatedly applying compressive loading, which simulated loading on cartilage in vivo. The released proteins were analyzed comprehensively by antibody array analyses and a quantitative proteomic analysis. For several proteins, the exact amounts released were determined by Luminex assays. The amount of active TGF-β that was released was determined by an assay using genetically-engineered HEK cells. The results of the antibody array and proteomic analyses revealed that various biologically active proteins are released from OA cartilage, particularly from DEG, by loading. The Luminex assay confirmed that several alarmins, complement proteins C3a and C5a, and several angiogenic proteins including FGF-1, FGF-2 and VEGF-A were released in greater amounts from DEG than from CONT. The HEK cell assay indicated that active TGF-β was released from DEG at biologically significant levels. These findings may be helpful in understanding the pathology of OA.

Topics: Cartilage, Articular; Humans; Knee Joint; Osteoarthritis; Proteomics; Transforming Growth Factor beta

Data from the Osteoarthritis Initiative shows that females who drink milk regularly have less joint cartilage loss and OA progression, but the biologic mechanism is unclear. Bovine milk is a rich source of extracellular vesicles (EVs), which are small phospholipid bilayer bound structures that facilitate intercellular communication. In this study, the authors aim to evaluate whether these EVs may have the capacity to protect cartilage from osteoarthritis patients, ex vivo, by directly effecting chondrocytes.. Human cartilage explants are exposed to cow's milk-derived EVs (CMEVs), which results in reduced sulfated glycosaminoglycan release and inhibition of metalloproteinase-1 expression. Incubation of articular chondrocytes with CMEVs also effectively reduces expression of cartilage destructive enzymes (ADAMTS5, MMPs), which play key roles in the disease progression. In part, these findings are attributed to the presence of TGFβ on these vesicles, and in addition, a possible role is reserved for miR-148a, which is functionally transferred by CMEVs.. These findings highlight the therapeutic potential of local CMEV delivery in osteoarthritic joints, where inflammatory and catabolic mediators are responsible for joint pathology. CMEVs are carriers of both TGFβ and miR-148a, two essential regulators for maintaining chondrocyte homeostasis and protection against cartilage destruction.

Topics: Animals; Cartilage, Articular; Cattle; Extracellular Vesicles; Female; Humans; MicroRNAs; Milk; Osteoarthritis; Transforming Growth Factor beta

Osteoarthritis (OA) is a cartilage degenerative disease commonly observed in the elderly population and is pathologically characterized by the degradation of the cartilage extracellular matrix (ECM). Matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs) are critical enzymes involved in the degradation of ECM. Olmesartan is an inhibitor of the angiotensin II receptor developed for the treatment of hypertension, and recent studies show that it exerts anti-inflammatory effects in arthritis. The present study aimed to investigate the mechanism of the protective effect of Olmesartan on cartilage ECM degradation. Interleukin-29 (IL-29) is a novel inflammatory mediator involved in the inflammation and degradation of cartilage in OA, and human T/C-28a2 cells treated with it were the inflammatory model

Topics: Anti-Inflammatory Agents; Cell Line; Cell Nucleus; Cell Proliferation; Cell Survival; Chondrocytes; Collagen Type II; Gene Expression Regulation; Humans; Imidazoles; Interleukins; Models, Biological; Osteoarthritis; Proteolysis; Tetrazoles; Transforming Growth Factor beta

Synovial fibrosis is a pathological process which contributes to joint pain and stiffness in several musculoskeletal disorders. Fucoidans, sulfated polysaccharides found in brown algae, have recently emerged as promising therapeutic agents. Despite the increasing amount of evidence suggesting the protective role of fucoidans in different experimental approaches of human fibrotic disorders, the effect of these sulfated polysaccharides on synovial fibrosis has not been investigated yet. By an in vitro experimental approach in fibroblast-like synoviocytes, we detected that fucoidans inhibit their differentiation into myofibroblasts with tumor cell-like characteristics and restore apoptosis. Composition and structure of fucoidan appear to be critical for the detected activity. Furthermore, protective effects of these sulfated polysaccharides are mediated by upregulation of nitric oxide production and modulation of TGF-β/smad pathway. Altogether, our results support the use of fucoidans as therapeutic compounds in the treatment of the fibrotic processes involved in rheumatic pathologies.

Topics: Aged; Aged, 80 and over; Apoptosis; Cell Movement; Cell Proliferation; Cells, Cultured; Extracellular Matrix Proteins; Female; Fibroblasts; Fibrosis; Humans; Male; Osteoarthritis; Phaeophyceae; Polysaccharides; Synoviocytes; Transforming Growth Factor beta

Osteoarthritis (OA) is a chronic joint disorder that causes cartilage degradation and subchondral bone abnormalities. Nangibotide, also known as LR12, is a dodecapeptide with considerable anti-inflammatory properties, but its significance in OA is uncertain. The aim of the study was to determine whether nangibotide could attenuate the progression of OA, and elucidate the underlying mechanism. In vitro experiments showed that nangibotide strongly inhibited TNF-α-induced osteogenic reduction, significantly enhanced osteoblast proliferation and prevented apoptosis in MC3T3-E1 cells. Male C57BL/6 J mice aged 2 months were randomly allocated to three groups: sham, ACLT, and ACLT with nangibotide therapy. Nangibotide suppressed ACLT-induced cartilage degradation and MMP-13 expression. MicroCT analysis revealed that nangibotide attenuated in vivo subchondral bone loss induced by ACLT. Histomorphometry results showed that nangibotide attenuated ACLT-induced osteoblast inhibition; TUNEL assays and immunohistochemical staining of cleaved-caspase3 further confirmed the in vivo anti-apoptotic effect of nangibotide on osteoblasts. Furthermore, we found that nangibotide exerted protective effects by suppressing TGF-β signaling mediated by Smad2/3 to restore coupled bone remodeling in the subchondral bone. In conclusion, the findings suggest that nangibotide might exert a protective effect on the bone-cartilage unit and maybe an alternative treatment option for OA.

Topics: Animals; Apoptosis; Cartilage, Articular; Disease Models, Animal; Lauric Acids; Male; Mice; Mice, Inbred C57BL; Oligopeptides; Osteoarthritis; Osteoblasts; Rhodamines; Transforming Growth Factor beta

During osteoarthritis (OA), hypertrophy-like chondrocytes contribute to the disease process. TGF-β's signaling pathways can contribute to a hypertrophy(-like) phenotype in chondrocytes, especially at high doses of TGF-β. In this study, we examine which transcription factors (TFs) are activated and involved in TGF-β-dependent induction of a hypertrophy-like phenotype in human OA chondrocytes. We found that TGF-β, at levels found in synovial fluid in OA patients, induces hypertrophic differentiation, as characterized by increased expression of

Topics: Chondrocytes; Humans; Hypertrophy; Osteoarthritis; Phenotype; Transforming Growth Factor beta; Transforming Growth Factors

The changed biomechanical environment of chondrocytes elicited by altered extracellular matrix is reported to accelerate the progression of OA. MicroRNAs (miRNAs or miRs) have emerged as major regulators in chondrocyte function. Hence, we explored effect of miR-23a/b-3p on OA in regulating chondrocyte growth. The medial meniscus and anterior cruciate ligaments of right knee was removed to induce a mouse model of OA. miR-23a/b-3p and Gremlin1 (Grem1) expressions in OA were determined by RT-qPCR. Dual luciferase reporter gene assay was conducted to assess their relationship in the context of OA. Loss- and gain-of-function assays were adopted to clarify their effects on OA by determining the release of pro-inflammatory proteins and the apoptosis of chondrocytes. RT-qPCR determined increased miR-23a/b-3p expression and decreased Grem1 expression in the setting OA. Inhibiting miR-23a/b-3p or overexpressing Grem1 activated transforming growth factor-β/solvated metal atom dispersed 3 (TGF-β/Smad) signaling to prevent OA development. Silencing Grem1 ablated suppressive effects of miR-23a/b-3p inhibitor on the release of pro-inflammatory proteins and the apoptosis of chondrocytes. To conclude, inhibition of miR-23a/b-3p delays OA progression through Grem1-mediated activation of TGF-β/Smad signaling, contributing to deepen understanding of the pathogenesis of OA.

Topics: Animals; Apoptosis; Chondrocytes; Intercellular Signaling Peptides and Proteins; Mice; MicroRNAs; Osteoarthritis; Transforming Growth Factor beta

Fibulin-3 is an extracellular matrix glycoprotein that is present in elastic tissue and involved in carcinoma development. Previous studies have indicated that fibulin-3 may affect skeletal development, cartilage, and osteoarthritis (OA). This study aims to investigate the function of fibulin-3 on chondrocytes under tumor necrosis factor alpha (TNF-α) stimulation and in murine OA models, and explore the possible mechanism. It was found that fibulin-3 was increased in the cartilage of OA models and in the chondrogenic cells ATDC5 stimulated by TNF-α. Fibulin-3 promoted the proliferation of ATDC5 cells both in the presence and absence of TNF-α. Moreover, overexpression of fibulin-3 suppressed the chondrogenic and hypertrophic differentiation of ATDC5 cells, while knockdown of fibulin-3 caused the opposite effect. Mechanistically, fibulin-3 partially suppressed the activation of TGF-β/Smad3 signaling by inhibiting the phosphorylation of Smad3. SIS3, a Smad3 inhibitor, decreased the chondrogenesis of articular cartilages in OA models, and partially reversed the chondrogenic differentiation of ATDC5 cells caused by knockdown of fibulin-3 in the presence of TNF-α. Furthermore, co-immunoprecipitation (Co-IP) showed that fibulin-3 could only interact with TGF-β type I receptor (TβRI), although overexpression of fibulin-3 reduced the protein levels of both TβRI and TβRII. In conclusion, this study indicates that fibulin-3 modulates the chondrogenic differentiation of ATDC5 cells in inflammation partially via TGF-β/Smad3 signaling pathway.

Topics: Animals; Calcium-Binding Proteins; Chondrocytes; Chondrogenesis; Mice; Osteoarthritis; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

This study is to investigate the role of 12-Epi-Napelline, a new alkaloid isolated from aconitum, in promoting the paracrine of Bone Mesenchymal Stem Cells (BMSCs) and the synergistic therapeutic effects on osteoarthritis.. We tested the cytotoxicity and optimization of 12-Epi-Napelline, and then simulated the osteoarthritis model in vitro damaging the chondrocytes by lipopolysaccharide (LPS) and RT-qPCR, Western blot and Immunofluorescence were used to detect the inflammatory factor IL-1β, COX-2, TNF-α, MMP-13 and anabolic cytokines of Col-2, BMP-2, TGF-β1 and Sox9 expression in chondrocytes after 12-Epi-Napelline treatment. Under the treatment of different time, Col-2, BMP-2, TGF-β1 and Sox9 expression in BMSCs were detected by RT-qPCR, Western blot, and Immunofluorescence. By establishing an osteoarthritis model in vivo, the anti-osteoarthritis effect of 12-Epi-Napelline or BMSCs was evaluated.. The results showed the expressions of IL-1β, COX-2, TNF-α, and MMP-13 were down-regulated in chondrocytes after 12-Epi-Napelline treatment, while the expression of Col-2, BMP-2, TGF-β1 and Sox9 were increased to normal chondrocytes. These increased expression also occurred in BMSCs. BMSCs had the trend of transforming into chondrocytes by regulating TGF-β signaling pathway under the treatment of 12-Epi-Napelline.. This study could confirm that 12-Epi-Napelline is not only effective in the treatment of osteoarthritis, but also can induce BMSCs to secrete growth factors that promote chondrocyte repair to help repair the damage caused by osteoarthritis.

Topics: Cell Differentiation; Cells, Cultured; Chondrocytes; Cyclooxygenase 2; Humans; Matrix Metalloproteinase 13; Mesenchymal Stem Cells; Osteoarthritis; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

In recent years, extracellular vesicles (EVs) secreted by mesenchymal stem cells (MSCs) have emerged as a potential cell-free therapy against osteoarthritis (OA). Thus, we investigated the therapeutic effects of EVs released by cytokine-primed umbilical cord-derived MSCs (UCMSCs) on osteoarthritic chondrocyte physiology. Priming UCMSCs individually with transforming growth factor beta (TGFβ), interferon alpha (IFNα), or tumor necrosis factor alpha (TNFα) significantly reduced the sorting of miR-181b-3p but not miR-320a-3p; two negative regulators of chondrocyte regeneration, into EVs. However, the EV treatment did not show any significant effect on chondrocyte proliferation. Meanwhile, EVs from both non-priming and cytokine-primed UCMSCs induced migration at later time points of measurement. Moreover, TGFβ-primed UCMSCs secreted EVs that could upregulate the expression of chondrogenesis markers (

Topics: Chondrocytes; Cytokines; Extracellular Vesicles; Humans; Mesenchymal Stem Cells; Osteoarthritis; Transforming Growth Factor beta; Umbilical Cord

Different opinions exist about the role of subchondral bone in osteoarthritis (OA), probably because subchondral bone has different effects on cartilage degeneration in OA induced by different pathologies. Animal studies to illustrate the role of subchondral bone in cartilage degeneration were mostly based on post-traumatic OA (PT-OA). Postmenopausal women experience a much higher occurrence of OA than similar-aged men. The physiological changes and pathogenesis of the osteochondral unit in ovariectomy-induced OA (OVX-OA) might be distinct from other types of OA.. The osteochondral alterations of post-traumatic OA (PT-OA) and OVX-OA at week 9 after surgery were compared. Then the alterations of osteochondral units in OVX-OA rats were tracked over time for the designed groups: Sham, OVX and OVX rats treated with estrogen (OVX+E). DXA, micro-CT, and histochemical staining were performed to observe alterations in osteochondral units.. Rapid cartilage degeneration and increased bone formation were observed in PT-OA, while only mild cartilage erosion and significant bone loss were observed in OVX-OA at week 9 after surgery. Subchondral bone degradation preceded cartilage degeneration by 6 weeks in OVX-OA. TGF-β expression was downregulated in the osteochondral unit of OVX rats. Estrogen supplementation inhibited subchondral bone loss, cartilage degradation and TGF-β expression decrease.. This research demonstrated the distinct behaviors of the osteochondral unit and the critical role of subchondral bone in early OVX-OA compared with PT-OA. Inhibiting subchondral bone catabolism at the early stage of OVX-OA could be an effective treatment for post-menopausal OA. Based on the results, estrogen supplementation and TGF-β modulation at the early stage are both potential therapies for post-menopausal OA.

Topics: Animals; Bone and Bones; Cartilage, Articular; Estrogens; Female; Osteoarthritis; Ovariectomy; Rats; Transforming Growth Factor beta; Wounds and Injuries

Aging is one of the primary factors influencing development of osteoarthritis, and the TGF-β pathway plays an important role in age-related osteoarthritis. Specifically, GDF15 phosphorylates SMAD2/3 in the TGF-β pathway to inhibit cardiomyocyte hypertrophy, and promote proliferation of chondrocytes. However, age-dependent changes in the level of GDF15 are unclear, as is whether GDF15 phosphorylates SMAD2/3 in the TGF-β pathway to promote proliferation of old chondrocytes. This study, therefore, sought to examine the effect of various GDF15 concentrations on old chondrocyte proliferation.. Serum and cartilage specimens of young adults and older adults were collected, and GDF15 expression was quantified. Human chondrocytes were then cultured following routine protocols, and different concentrations of recombinant human GDF15 or pSMAD2 inhibitor were added into the culture medium. After 48 h of culturing, the proliferation of chondrocytes was detected by EdU, and the expression MMP13, SMAD2, and pSMAD2 was detected in chondrocytes via western blot and qRT-PCR analysis.. The GDF15 content in serum and cartilage of young adults was higher than that of older adults (p < 0.05). The number of EdU-positive cells in the experimental group (containing recombinant human GDF15) was higher than that in the control group (medium only) (p < 0.05). Compared with the control group, chondrocytes in the experimental group showed increased pSMAD2 and type II collagen content (p < 0.05) and decreased MMP13 (p < 0.05), with no significant difference in SMAD2 content (p > 0.05). Moreover, no significant differences were observed between the control group and the TGF-β signaling inhibitor group. The gene expression level of each index was consistent with the protein expression level.. The GDF15 content of serum and cartilage in young adults is higher than in older adults, and GDF15 functions to promote the proliferation of chondrocytes by phosphorylating SMAD2 in older individuals.

Topics: Aged; Cell Proliferation; Chondrocytes; Gene Expression; Growth Differentiation Factor 15; Humans; Osteoarthritis; Smad2 Protein; Transforming Growth Factor beta

Osteoarthritis (OA) is an age-related disease, which is mainly treated with oral, topical, and/or intra-articular options to relieve symptoms and lack of specific treatment measures. Fibroblasts (FLS) are crucial cells in joint inflammation and destruction. Cellular senescence plays an important role during OA pathogenesis and senescent cells exhibit cell-cycle arrest and senescence-associated secretory phenotype (SASP). SRY-related HMG-box 4 (SOX4) is a contributing factor during many developmental processes and is elevated in inflamed synovium than in noninflamed synovium from arthritis patients. This study was designed to investigate whether SOX4 participate in the pathogenesis of OA by affecting FLS senescence and explore the internal mechanism. Firstly, we found that FLS cells exhibited more cellular senescence in OA compared with control group. We also verified the role of reactive oxygen species (ROS)/TGF-β signal in the induction of OA-FLS senescence. During the exploration of SOX4 in cell senescence, the results indicated that SOX4 activation promotes cell senescence and SASP of OA-FLS. Apart from that, we also confirmed that SOX4, regulated by ROS/TGF-β signal, was critical transcription factor associated with OA-FLS senescence. Therefore, SOX4 is likely to be a novel therapeutic target and early diagnostic marker during OA pathogenesis.

Topics: Cells, Cultured; Cellular Senescence; Fibroblasts; Humans; Osteoarthritis; Reactive Oxygen Species; Signal Transduction; SOXC Transcription Factors; Transforming Growth Factor beta

Transforming growth factor β (TGFβ) signaling plays a complex tissue-specific and nonlinear role in osteoarthritis (OA). This study was conducted to determine the osteocytic contributions of TGFβ signaling to OA.. In all contexts, independent of sex, genotype, or medial or lateral joint compartment, increased SBP thickness and SBP sclerostin expression were spatially associated with cartilage degeneration. Male TβRII. Our results provide new evidence that osteocytic TGFβ signaling is required for a mechanosensitive response to injury, and that osteocytes control SBP homeostasis to maintain cartilage health, identifying osteocytic TGFβ signaling as a novel therapeutic target for OA.

Topics: Adaptor Proteins, Signal Transducing; Animals; Bone and Bones; Cartilage, Articular; Female; Hindlimb; Homeostasis; Male; Mechanotransduction, Cellular; Medial Collateral Ligament, Knee; Menisci, Tibial; Mice; Mice, Knockout; Osteoarthritis; Osteocytes; Receptor, Transforming Growth Factor-beta Type II; Sex Factors; Signal Transduction; Transforming Growth Factor beta; X-Ray Microtomography

Osteoarthritis (OA) is the most common joint disease. The surface of joint cartilage is a defensive and first affected structure of articular cartilage (AC) during the pathogenesis of OA. Alk5 signaling is critical for maintaining AC homeostasis, however, the role and underlying mechanism for the involvement of Alk5 signaling in the phenotypes of articular cartilage stem cells (ACSCs) at the surface of AC is still unclear. The role of Alk5 in OA development was explored using an ACSCs-specific Alk5-deficient (cKO) mouse model. Alterations in cartilage structure were evaluated histologically. Senescence was detected by SA-β-gal, while reactive oxygen species (ROS), MitoTracker, and LysoTracker staining were used to detect changes related to senescence. In addition, mice were injected intra-articularly with ganciclovir to limit the detrimental roles of senescent cells (SnCs). Alk5 cKO mice showed a decreased number of the slow-cell cycle cells and less lubricant secretion at the surface accompanied with drastically accelerated cartilage degeneration under ageing and surgically induced OA conditions. Further studies showed that Alk5 deficient ACSCs exhibited senescence-like manifestations including decreased proliferation and differentiation, more SA-β-gal-positive cells and ROS production, as well as significantly swollen mitochondria and lysosome breakdown. We further found that local limitation of the detrimental roles of SnCs can attenuate the development of posttraumatic OA. Taken together, our findings suggest that Alk5 signaling acts as an important regulator of the SnCs in the superficial layer during AC maintenance and OA initiation.

Topics: Animals; Arthritis, Experimental; Cartilage, Articular; Cellular Senescence; Mice; Mice, Knockout; Osteoarthritis; Receptor, Transforming Growth Factor-beta Type I; Signal Transduction; Stem Cells; Transforming Growth Factor beta

To investigate the role of LncRNA PVT1 (plasmacytoma variant translocation 1) in hyperglycemia-triggered cartilage damage using the diabetic osteoarthritis (OA) mice model.. Streptozotocin (STZ) was used to induce mouse diabetes. Knee OA model was induced through transection of anterior cruciate ligament (ACLT). Severity of arthritis was assessed histologically by Safranin O-Fast Green Staining using Mankin Scores. LncRNA PVT1 and miR-146a were detected by real-time polymerase chain reaction (PCR) in cartilage tissue. Moreover, the interaction among PVT1, miR-146a, and SMAD4 was examined by luciferase reporter assays. Mice were injected intra-articularly with ad-siRNA-PVT1 and ad-siRNA scramble control. Articular concentrations of TNF-α, IL-1, IL-6 and TGF-β1 were determined using enzyme-linked immunosorbent assay. Levels of type II Collagen (COL2A1), TGF-β1, p-SMAD2, SMAD2, p-SMAD3, SMAD3, SMAD4 and nuclear SMAD4 were detected by western blot analysis.. PVT1 expression was significantly increased, whereas miR-146a was markedly decreased in diabetic OA mice than in non-diabetic OA and control. Increased PVT1 expression in diabetic OA mice was significantly associated with Mankin score and reduced miR-146a as well as Collagen alpha-1(II) (COL2A1) expressions. In vivo, intra-articular injection of ad-siRNA-PVT1 efficiently increased miR-146a and COL2A1 expressions, alleviated joint inflammation, decreased the expression of pro-inflammatory mediators, and suppressed TGF-β/SMAD4 pathway in diabetic OA mice.. Our results demonstrate LncRNA PVT1 is involved in cartilage degradation in diabetic OA and correlated with disease severity. Efficiency of ad-siRNA-PVT1 in controlling joint inflammation in diabetic OA mice is associated with the suppression of the expression of miR-146a, pro-inflammatory cytokines and activation of TGF-β/SMAD4 pathway.

Topics: Adenoviridae; Animals; Base Sequence; Cartilage, Articular; Collagen Type II; Diabetes Mellitus, Experimental; Down-Regulation; HEK293 Cells; Humans; Hyperglycemia; Matrix Metalloproteinase 13; Matrix Metalloproteinase 3; Mice, Inbred C57BL; MicroRNAs; Osteoarthritis; RNA, Long Noncoding; RNA, Small Interfering; Severity of Illness Index; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

Osteoarthritis (OA) is a multifactorial disease with huge phenotypic heterogeneity. The disease affects all tissues in the joint, and the loss of articular cartilage is its hallmark. The main biochemical components of the articular cartilage are type II collagen, aggrecan, and water. Transforming growth factor-beta (TGF-β) signaling is one of the signaling pathways that maintains the healthy cartilage. However, the two subpathways of the TGF-β signaling-TGF-β and bone morphogenetic proteins (BMP) subpathways, lose their balance in OA, resulting an increased expression of cartilage degradation enzymes including matrix metallopeptidase 13 (MMP13), cathepsin B (CTSB), and cathepsin K (CTSK) and a decreased expression of aggrecan (ACAN). Thus, restoring the balance of two subpathways might provide a new avenue for treating OA patients. Further, metabolic changes are seen in OA and can be used to distinguish different subtypes of OA patients. Metabolomics studies showed that at least three endotypes of OA can be distinguished: 11% of OA patients are characterized by an elevated blood butyryl carnitine, 33% of OA patients have significant reduced arginine concentration, and 56% with metabolic alteration in phospholipid metabolism. While these findings need to be confirmed, they are promising personalized medicine tools for OA management.

Topics: Biomarkers; Bone Morphogenetic Proteins; Cartilage; Humans; Nuclear Export Signals; Osteoarthritis; Transforming Growth Factor beta

The transforming growth factor-β (TGF-β) signaling pathway plays an essential role in maintaining homeostasis in joints affected by osteoarthritis (OA). However, the specific mechanism of non-SMAD and classical SMAD signaling interactions is still unclear, which needs to be further explored.. In ATDC5 cells, USP15 overexpression and knockout were performed using the transfected lentivirus USP15 and Crispr/Cas9. Western blotting and immunofluorescence staining were used to test p-SMAD2 and cartilage phenotype-related molecular markers. In rat OA models, immunohistochemistry, hematoxylin and eosin (HE)/Safranin-O fast green staining, and histology were used to examine the regulatory activity of USP15 in TGF-β/SMAD2 signaling and the cartilage phenotype. Then, ERK2 overexpression and knockout were performed. The expressions of USP15, p-SMAD2, and the cartilage phenotype were evaluated in vitro and in vivo. To address whether USP15 is required for ERK2 and TGF-β/SMAD2 signaling, we performed rescue experiments in vitro and in vivo. Immunoprecipitation and deubiquitination assays were used to examine whether USP15 could bind to ERK2 and affect the deubiquitination of ERK2. Finally, whether USP15 regulates the level of p-ERK1/2 was evaluated by western blotting, immunofluorescence staining, and immunohistochemistry in vitro and in vivo.. Our results indicated that USP15 stimulated TGF-β/SMAD2 signaling and the cartilage phenotype. Moreover, ERK2 required USP15 to influence TGF-β/SMAD2 signaling for regulating the cartilage phenotype in vivo and in vitro. And USP15 can form a complex with ERK2 to regulate ubiquitination of ERK2. Interestingly, USP15 did not regulate the stability of ERK2 but increased the level of p-ERK1/2 to further enhance the TGF-β/SMAD2 signaling pathway.. Taken together, our study revealed positive feedback regulation between USP15 and ERK2, which played a critical role in TGF-β/SMAD2 signaling to inhibit OA progression. Therefore, this specific mechanism can guide the clinical treatment of OA.

Topics: Animals; Cartilage, Articular; Chondrocytes; Endopeptidases; Feedback, Physiological; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Osteoarthritis; Rats; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta

Our incomplete understanding of osteoarthritis (OA) pathogenesis has significantly hindered the development of disease-modifying therapy. The functional relationship between subchondral bone (SB) and articular cartilage (AC) is unclear. Here, we found that the changes of SB architecture altered the distribution of mechanical stress on AC. Importantly, the latter is well aligned with the pattern of transforming growth factor beta (TGFβ) activity in AC, which is essential in the regulation of AC homeostasis. Specifically, TGFβ activity is concentrated in the areas of AC with high mechanical stress. A high level of TGFβ disrupts the cartilage homeostasis and impairs the metabolic activity of chondrocytes. Mechanical stress stimulates talin-centered cytoskeletal reorganization and the consequent increase of cell contractile forces and cell stiffness of chondrocytes, which triggers αV integrin-mediated TGFβ activation. Knockout of αV integrin in chondrocytes reversed the alteration of TGFβ activation and subsequent metabolic abnormalities in AC and attenuated cartilage degeneration in an OA mouse model. Thus, SB structure determines the patterns of mechanical stress and the configuration of TGFβ activation in AC, which subsequently regulates chondrocyte metabolism and AC homeostasis.

Topics: Animals; Bone and Bones; Cartilage, Articular; Cell Line; Chondrocytes; Cytoskeleton; Homeostasis; Humans; Integrin alphaV; Mice; Mice, Inbred C57BL; Mice, Transgenic; Osteoarthritis; Signal Transduction; Stress, Mechanical; Talin; Transforming Growth Factor beta

Osteoarthritis (OA) is a serious joint disease with no disease-modifying medical treatment. To develop treatments targeting synovium, we must improve our understanding of the effects of OA-related changes in synovial physiology on joint tissue outcomes. The aim of this study was to investigate the effects of synovial pathology due to post-traumatic OA (PTOA) on articular chondrocyte physiology.. We first developed and validated a novel joint tissue co-culture system to model the biological interactions between synovium and articular chondrocytes. Whole-joint synovial tissue from a surgical rat model of PTOA vs sham and surgical-naïve controls was placed into a co-culture system with adult primary articular chondrocytes (n = 4-5). The effects of PTOA synovium on chondrocyte anabolic, inflammatory, and catabolic gene expression and sulfated glycosaminoglycan (sGAG) secretion and aggrecan synthesis were tested, and results from early and later stages of PTOA development were compared.. Synovial injury by arthrotomy (sham surgery) alone decreased primary chondrocyte expression of genes including Col2a1 (0.36 ± 0.15-fold) and Acan (0.41 ± 0.28-fold). Early PTOA synovium rescued the suppression of Acan, induced increased sGAG secretion (3.94 ± 0.44 μg/mL vs surgery-naïve 2.41 ± 0.55 and sham 2.92 ± 0.73 μg/mL controls), and upregulated Mmp3 (3.73 ± 2.62-fold) and Prg4 (4.93 ± 4.29-fold). These effects were lost with later stage PTOA synovium.. Early PTOA synovium induces transient anabolic responses in articular chondrocytes rather than pro-inflammatory responses that would require inhibition. These results suggest that PTOA synovium plays at least a partially protective role and that loss of these protective effects may contribute to PTOA progression.

Topics: Animals; Cartilage, Articular; Chemokine CCL2; Chondrocytes; Coculture Techniques; Disease Models, Animal; Osteoarthritis; Proteoglycans; Rats, Sprague-Dawley; Synovial Membrane; Transforming Growth Factor beta

Chondrocytes in mice developing osteoarthritis (OA) exhibit an aberrant response to the secreted cytokine transforming growth factor (TGF)-β, consisting in a potentiation of intracellular signaling downstream of the transmembrane type I receptor kinase activin receptor-like kinase (ALK)1 against canonical TGF-β receptor ALK5-mediated signaling. Unfortunately, the underlying mechanisms remain elusive. In order to identify novel druggable targets for OA, we aimed to investigate novel molecules regulating the ALK1/ALK5 balance in OA chondrocytes. We performed gene expression analysis of TGF-β signaling modulators in joints from three different mouse models of OA and found an upregulated expression of the TGF-β co-receptor Cripto (Tdgf1), which was validated in murine and human cartilage OA samples at the protein level. In vitro and ex vivo, elevated expression of Cripto favors the hypertrophic differentiation of chondrocytes, eventually contributing to tissue calcification. Furthermore, we found that Cripto participates in a TGF-β-ALK1-Cripto receptor complex in the plasma membrane, thereby inducing catabolic SMAD1/5 signaling in chondrocytes. In conclusion, we demonstrate that Cripto is expressed in OA and plays a functional role promoting chondrocyte hypertrophy, thereby becoming a novel potential therapeutic target in OA, for which there is no efficient cure or validated biomarker. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Topics: Animals; Chondrocytes; Epidermal Growth Factor; GPI-Linked Proteins; Humans; Hypertrophy; Intercellular Signaling Peptides and Proteins; Membrane Glycoproteins; Mice; Neoplasm Proteins; Osteoarthritis; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Osteoarthritis is the most prevalent musculoskeletal disease, affecting 250 million people worldwide.

Topics: Aged; Aggrecans; Cartilage, Articular; Heparitin Sulfate; Humans; Osteoarthritis; Sodium; Transforming Growth Factor beta

To identify the role of misshapen/NIK-related kinase (MINK1) in age-related Osteoarthritis (OA) and injury-induced OA, and the effects of enhanced TGFβ signaling in these progresses.. The effect of MINK1 was analyzed with MINK1 knock out (Mink1-/-) mice and C57BL/6J mice. OA progress was studied in age-related OA and instability-associated OA (destabilization of the medial meniscus, DMM) models. The murine knee joint was evaluated through histological staining, Osteoarthritis Research Society International (OARSI) scores, immunohistochemistry, and μCT analysis. Primary chondrocytes were isolated from wild type and Mink1-/- mice and subjected to osteogenic induction and Western blot analysis.. MINK1 is highly expressed during cartilage development and in normal cartilage. Mink1-/- mice displayed markedly lower OARSI scores, aggrecan degradation neoepitope positive cells and increased Safranin O and pSMAD2 staining in aging-related OA model. However, in injury-induced OA, loss of MINK1 accelerates extracellular matrix (ECM) destruction, osteophyte formation, and subchondral bone sclerosis. Accelerated subchondral bone remodeling in Mink1-/- mice was accompanied with increased numbers of nestin-positive mesenchymal stem cells (MSCs) and osterix-positive osteoprogenitors. pSMAD2 staining was increased in the subchondral bone marrow of Mink1-/- mice and overexpression of MINK1 inhibited SMAD2 phosphorylation in vitro.. This study shows for the first time that activation of TGFβ/SMAD2 by MINK1 deficiency plays opposite roles in aging-related and injury-induced OA. MINK1 deficiency protects cartilage from degeneration in aging joints through increased SMAD2 activation in chondrocytes, while accelerating OA progress in injury-induced model through enhanced osteogenesis of MSCs in the subchondral bone. These findings provide insights for developing precision OA therapeutics targeting TGFβ/SMAD2 signaling.

Topics: Animals; Blotting, Western; Cells, Cultured; Chondrocytes; Disease Models, Animal; Disease Progression; Joints; Male; Mice, Inbred C57BL; Mice, Knockout; Osteoarthritis; Osteogenesis; Protein Serine-Threonine Kinases; Signal Transduction; Transforming Growth Factor beta; X-Ray Microtomography

TGFβ is a key player in cartilage homeostasis and OA pathology. However, few data are available on the role of TGFβ signalling in the different OA phenotypes. Here, we analysed the TGFβ pathway by transcriptomic analysis in six mouse models of OA.. We have brought together seven expert laboratories in OA pathophysiology and, used inter-laboratories standard operating procedures and quality controls to increase experimental reproducibility and decrease bias. As none of the available OA models covers the complexity and heterogeneity of the human disease, we used six different murine models of knee OA: from post-traumatic/mechanical models (meniscectomy (MNX), MNX and hypergravity (HG-MNX), MNX and high fat diet (HF-MNX), MNX and seipin knock-out (SP-MNX)) to aging-related OA and inflammatory OA (collagenase-induced OA (CIOA)). Four controls (MNX-sham, young, SP-sham, CIOA-sham) were added. OsteoArthritis Research Society International (OARSI)-based scoring of femoral condyles and ribonucleic acid (RNA) extraction from tibial plateau samples were done by single operators as well as the transcriptomic analysis of the TGFβ family pathway by Custom TaqMan® Array Microfluidic Cards.. The transcriptomic analysis revealed specific gene signatures in each of the six models; however, no gene was deregulated in all six OA models. Of interest, we found that the combinatorial Gdf5-Cd36-Ltbp4 signature might discriminate distinct subgroups of OA: Cd36 upregulation is a hallmark of MNX-related OA while Gdf5 and Ltbp4 upregulation is related to MNX-induced OA and CIOA.. These findings stress the OA animal model heterogeneity and the need of caution when extrapolating results from one model to another.

Topics: Animals; Arthritis, Experimental; CD36 Antigens; Collagenases; Diet, High-Fat; Disease Models, Animal; Gene Expression Profiling; Growth Differentiation Factor 5; GTP-Binding Protein gamma Subunits; Hypergravity; Latent TGF-beta Binding Proteins; Meniscectomy; Metabolic Syndrome; Mice; Mice, Knockout; Obesity; Osteoarthritis; Transcriptome; Transforming Growth Factor beta

Transglutaminase 2 (TG2), also known as tissue transglutaminase, is a calcium-dependent enzyme that has a variety of intracellular and extracellular substrates. TG2 not only increases in osteoarthritis (OA) tissue but also affects the progression of OA. However, it is still unclear how TG2 affects cartilage degradation in OA at the molecular level. Surgically induced OA lead to an increase of TG2 in the articular cartilage and growth plate, and it was dependent on TGFβ1 in primary chondrocytes. The inhibition of TG2 enzymatic activity with intra-articular injection of ZDON, the peptide-based specific TG2 inhibitor, ameliorated the severity of surgically induced OA as well as the expression of MMP-3 and MMP-13. ZDON attenuated MMP-3 and MMP-13 expression in TGFβ- and calcium ionophore-treated chondrocytes in a Runx2-independent manner. TG2 inhibition with ZDON suppressed canonical Wnt signaling through a reduction of β-catenin, which was mediated by ubiquitination-dependent proteasomal degradation. In addition, TG2 activation by a calcium ionophore enhanced the phosphorylation of AMPK and FoxO3a and the nuclear translocation of FoxO3a, which was responsible for the increase in MMP-13. In conclusion, TG2 plays an important role in the pathogenesis of OA as a major catabolic mediator that affects the stability of β-catenin and FoxO3a-mediated MMP-13 production.

Topics: Animals; beta Catenin; Calcium; Cartilage, Articular; Cells, Cultured; Forkhead Box Protein O3; Growth Plate; GTP-Binding Proteins; Male; Matrix Metalloproteinase 13; Matrix Metalloproteinase 3; Mice; Mice, Inbred C57BL; NF-kappa B; Osteoarthritis; Patient Acuity; Protein Glutamine gamma Glutamyltransferase 2; Transforming Growth Factor beta; Transglutaminases; Wnt Signaling Pathway

Topics: Animals; Apoptosis; Cartilage; Cell Proliferation; Chondrocytes; Cyclin-Dependent Kinases; Disease Models, Animal; Disease Progression; Down-Regulation; Extracellular Matrix; Humans; In Situ Hybridization, Fluorescence; Male; MicroRNAs; Osteoarthritis; Rabbits; RNA, Circular; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta

Human osteoarthritis cartilage contains chondrocytes (OAC) and mesenchymal stromal cells (OA-MSC). Here, we found that TGF-β had different effects on OA-MSC and OAC, and revealed its lateral signaling mechanism in OA. RNAseq analysis indicated that OA-MSC expressed the same level of Bone Morphogenetic Protein (BMP) Receptor-1A as OAC but only 1/12 of Transforming Growth Factor beta (TGF-β) Receptor-1. While TGF-β specifically activated SMAD2 in OAC, it also activated BMP signaling-associated SMAD1 in OA-MSC. While TGF-β stimulated chondrogenesis in OAC, it induced hypertrophy, mineralization, and MMP-13 in OA-MSC. Inhibiting TGF-βR1 suppressed MMP-13 in OA-MSC but stimulated it in OAC. In contrast, by specifically targeting BMPR1A/ACVR1 in both cell types, LDN193189 inhibits cartilage degeneration through suppressing hypertrophy and MMP-13 in a mouse osteoarthritis model. Thus, LDN193189, a drug under development to inhibit constitutive BMP signaling during heterotopic ossification, may be re-purposed for OA treatment.

Topics: Animals; Cartilage, Articular; Cells, Cultured; Chondrocytes; Chondrogenesis; Humans; Hypertrophy; Male; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; Osteoarthritis; Receptor, Transforming Growth Factor-beta Type I; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta

Transforming growth factor-β (TGF-β) signaling is a critical regulator for articular cartilage tissue maintenance and chondrocyte homeostasis. Nonetheless, the regulatory networks and downstream signaling pathways that govern the chondroprotective function of TGF-β in the context of osteoarthritis (OA) are not fully defined. Recent studies reveal that mice with postnatal deletion of triple forkhead box class Os (FoxOs) (1, 3, and 4) spontaneously develop OA-like pathologies. The OA phenotype largely recapitulates that observed in mice with loss of TGF-βR2. In the present study, we investigated the role of FoxOs as downstream mediators of TGF-β signaling and define their role in articular cartilage homeostasis. Among the three FoxOs (1, 3, and 4), TGF-β signaling exclusively regulates FoxO1 in a TGF-β activated kinase 1 (TAK1)-dependent manner. Furthermore, FoxO1 was genetically ablated in mice in a tissue-specific manner in articular cartilage or overexpressed in adult cartilage immediately followed by meniscal/ligament injury (MLI). Histological and microcomputed tomography (micro-CT) analyses demonstrated that loss of FoxO1 postnatally in articular cartilage leads to OA-like pathologies, and gain of FoxO1 in adult cartilage has both preventative and therapeutic effects on surgically induced OA. Mechanistically, FoxO1 was found to maintain articular chondrocyte homeostasis through induction of anabolic and autophagy-related gene expressions. Importantly, overexpression of FoxO1 markedly rescued the OA phenotypes caused by deficiency in TGF-β signaling in chondrocytes. Our study identifies that TGF-β/TAK1-FoxO1 is a key signaling cascade in regulation of articular cartilage autophagy and homeostasis and is a potentially important therapeutic target for OA-like joint diseases.

Topics: Animals; Autophagy; Biomarkers; Cartilage, Articular; Chondrocytes; Disease Models, Animal; Disease Susceptibility; Forkhead Box Protein O1; Forkhead Transcription Factors; Homeostasis; MAP Kinase Kinase Kinases; Mice; Mice, Knockout; Models, Biological; Osteoarthritis; Reactive Oxygen Species; Signal Transduction; Transforming Growth Factor beta; X-Ray Microtomography

Adipose-derived mesenchymal stromal cells (Ad-MSCs) are a promising tool for articular cartilage repair and regeneration. However, the terminal hypertrophic differentiation of Ad-MSC-derived cartilage is a critical barrier during hyaline cartilage regeneration. In this study, we investigated the role of matrilin-3 in preventing Ad-MSC-derived chondrocyte hypertrophy in vitro and in an osteoarthritis (OA) destabilization of the medial meniscus (DMM) model. Methacrylated hyaluron (MAHA) (1%) was used to encapsulate and make scaffolds containing Ad-MSCs and matrilin-3. Subsequently, the encapsulated cells in the scaffolds were differentiated in chondrogenic medium (TGF-β, 1-14 days) and thyroid hormone hypertrophic medium (T3, 15-28 days). The presence of matrilin-3 with Ad-MSCs in the MAHA scaffold significantly increased the chondrogenic marker and decreased the hypertrophy marker mRNA and protein expression. Furthermore, matrilin-3 significantly modified the expression of TGF-β2, BMP-2, and BMP-4. Next, we prepared the OA model and transplanted Ad-MSCs primed with matrilin-3, either as a single-cell suspension or in spheroid form. Safranin-O staining and the OA score suggested that the regenerated cartilage morphology in the matrilin-3-primed Ad-MSC spheroids was similar to the positive control. Furthermore, matrilin-3-primed Ad-MSC spheroids prevented subchondral bone sclerosis in the mouse model. Here, we show that matrilin-3 plays a major role in modulating Ad-MSCs' therapeutic effect on cartilage regeneration and hypertrophy suppression.

Topics: Animals; Bone Morphogenetic Protein 2; Cell Differentiation; Cell Proliferation; Chondrocytes; Chondrogenesis; Humans; Hyaline Cartilage; Hyaluronic Acid; Hypertrophy; Matrilin Proteins; Mesenchymal Stem Cells; Mice; Osteoarthritis; Regeneration; Spheroids, Cellular; Tissue Scaffolds; Transforming Growth Factor beta

Rheumatoid arthritis (RA) and osteoarthritis (OA) are common rheumatic disorders that primarily involve joints. The inflammation of the synovium can be observed in both of the two diseases. Synovial fibroblasts (SFs) play an important role in the inflammatory process of the synovium. The functional states of synovial fibroblasts are heterogeneous, and the detailed transition process of their functional states is still unclear. By using transcriptomic data of SFs at a single-cell level, we found a similar transition process for SFs in RA and OA. We also identified the potential regulatory effects of the WNT signaling pathway, the TGF-

Topics: Arthritis, Rheumatoid; Disease Progression; Fibroblasts; Genes, erbB; Humans; Osteoarthritis; RNA-Seq; Single-Cell Analysis; Synovial Membrane; Transforming Growth Factor beta; Wnt Signaling Pathway

MicroRNAs (miRNAs, miR) play a key role in the pathogenesis of osteoarthritis (OA). Few studies have examined the regulatory role of P21-activated kinases (PAKs), a family of serine/threonine kinases, in OA. The aim of this study was to determine whether miR-455-3p can regulate cartilage degeneration in OA by targeting PAK2. MiR-455-3p knockout mice showed significant degeneration of the knee cartilage. MiR-455-3p expression increased and PAK2 expression decreased in the late stage of human adipose-derived stem cell (hADSC) chondrogenesis and in chondrocytes affected by OA. Furthermore, in both miR-455-3p-overexpressing chondrocytes and PAK2-suppressing chondrocytes, cartilage-specific genes were upregulated, and hypertrophy-related genes were downregulated. A luciferase reporter assay confirmed that miR-455-3p regulates PAK2 expression by directly targeting the 3'-untranslated regions (3'UTRs) of PAK2 mRNA. IPA-3, a PAK inhibitor, inhibited cartilage degeneration due to OA. Moreover, suppressing PAK2 promoted R-Smad activation in the TGF/Smad signaling pathway in chondrocytes. Altogether, our results suggest that miR-455-3p promotes TGF-β/Smad signaling in chondrocytes and inhibits cartilage degeneration by directly suppressing PAK2. These results thus indicate that miR-455-3p and PAK2 are novel potential therapeutic agents and targets, respectively, for the treatment of OA.

Topics: Animals; Cartilage; Chondrocytes; Chondrogenesis; Disease Models, Animal; Humans; Mice; Mice, Knockout; MicroRNAs; Osteoarthritis; p21-Activated Kinases; Signal Transduction; Transforming Growth Factor beta

Localized delivery of drugs into an osteoarthritic cartilaginous lesion does not yet exist, which limits pharmaceutical management of osteoarthritis (OA). High-intensity focused ultrasound (HIFU) provides a means to actuate matter from a distance in a non-destructive way. In this study, we aimed to deliver methylene blue locally into bovine articular cartilage in vitro. HIFU-treated samples (n = 10) were immersed in a methylene blue (MB) solution during sonication (f = 2.16 MHz, peak-positive-pressure = 3.5 MPa, mechanical index = 1.8, pulse repetition frequency = 3.0 kHz, cycles per burst: 50, duty cycle: 7%). Adjacent control 1 tissue (n = 10) was first pre-treated with HIFU followed by immersion into MB; adjacent control 2 tissue (n = 10) was immersed in MB without ultrasound exposure. The MB content was higher (p < 0.05) in HIFU-treated samples all the way to a depth of 600 µm from AC surface when compared to controls. Chondrocyte viability and RNA expression levels associated with cartilage degeneration were not different in HIFU-treated samples when compared to controls (p > 0.05). To conclude, HIFU delivers molecules into articular cartilage without major short-term concerns about safety. The method is a candidate for a future approach for managing OA.

Topics: Animals; Cartilage, Articular; Cattle; Cell Survival; Chondrocytes; High-Intensity Focused Ultrasound Ablation; Matrix Metalloproteinases; Methylene Blue; Osteoarthritis; Transforming Growth Factor beta

Upregulation of calcium/calmodulin-dependent kinase II (CaMKII) is implicated in the pathogenesis of osteoarthritis (OA) and reactivation of articular cartilage hypertrophy. However, direct inhibition of CaMKII unexpectedly augmented symptoms of OA in animal models. The role of CaMKII in OA remains unclear and requires further investigation.. Analysis of CaMKII expression was performed in normal human and OA articular chondrocytes, and signaling mechanisms were assessed in articular, fetal and Pluripotent Stem Cell (PSC)-derived human chondrocytes using pharmacological (KN93), peptide (AC3-I) and small interfering RNA (siRNA) inhibitors of CaMKII.. Expression levels of phospho-CaMKII (pCaMKII) were significantly and consistently increased in human OA specimens. BMP2/4 activated expression of pCaMKII as well as COLII and COLX in human adult articular chondrocytes, and also increased the levels and nuclear localization of SMADs1/5/8, while TGFβ1 showed minimal or no activation of the chondrogenic program in adult chondrocytes. Targeted blockade of CaMKII with specific siRNAs decreased levels of pSMADs, COLII, COLX and proteoglycans in normal and OA adult articular chondrocytes in the presence of both BMP4 and TGFβ1. Both human fetal and PSC-derived chondrocytes also demonstrated a decrease of chondrogenic differentiation in the presence of small molecule and peptide inhibitors of CaMKII. Furthermore, immunoprecipitation for SMADs1/5/8 or 2/3 followed by western blotting for pCaMKII showed direct interaction between SMADs and pCaMKII in primary chondrocytes.. Current study demonstrates a direct role for CaMKII in TGF-β and BMP-mediated responses in primary and PSC-derived chondrocytes. These findings have direct implications for tissue engineering of cartilage tissue from stem cells and therapeutic management of OA.

Topics: Aged; Bone Morphogenetic Protein 4; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cartilage, Articular; Cell Differentiation; Cells, Cultured; Chondrocytes; Collagen Type II; Collagen Type X; Extracellular Matrix Proteins; Female; Humans; Male; Middle Aged; Osteoarthritis; Phosphorylation; Pluripotent Stem Cells; Signal Transduction; Smad Proteins, Receptor-Regulated; Transforming Growth Factor beta; Up-Regulation

Tissue inhibitor of metalloproteinase (TIMP)-3 is a natural inhibitor of a range of enzymes that degrade connective tissue and are involved in the pathogenesis of conditions such as arthritis and cancer. We describe here the engineering of TIMP-3 using a novel drug-delivery system known as the 'LAP technology'. This involves creating therapeutic proteins in fusion with the latency-associated peptide (LAP) from the cytokine TGF-? to generate proteins that are biologically inactive until cleavage of the LAP to release the therapy. LAP-TIMP-3 was successfully expressed in mammalian cells and the presence of the LAP resulted in a 14-fold increase in the quantity of recombinant TIMP-3 produced. LAP-TIMP-3 was latent until release from the LAP by treatment with matrix metalloproteinase when it could inhibit proteases of the adamalysins and adamalysins with thrombospondin motifs families, but not matrix metalloproteinases, indicating that this version of TIMP-3 is a more specific inhibitor than the native protein. There was sufficient protease activity in synovial fluid from human joints with osteoarthritis to release TIMP-3 from the LAP fusion. These results demonstrate the potential for development of TIMP-3 as a novel therapy for conditions where upregulation of catabolic enzymes are part of the pathology.

Topics: Aged; Aged, 80 and over; Animals; Cartilage; Cattle; Cytokines; Female; Humans; Inflammation; Male; Middle Aged; Osteoarthritis; Peptides; Protein Precursors; Recombinant Fusion Proteins; Recombinant Proteins; Synovial Fluid; Tissue Inhibitor of Metalloproteinase-3; Transforming Growth Factor beta

Osteoarthritis (OA) is a degenerative joint disease characterized by the degradation of joint cartilage, the formation of osteophyma at joint margins, and synovial changes. Whereas lesions of the joint cartilage were the key point of the research and treatment of osteoarthritis before, a recent study showed that the synovium plays a crucial role in the pathological progress of OA. The inflammatory environment in the joints of OA patients always results in the overactivation of fibroblast-like synoviocytes (FLSs), which produce a multitude of inflammatory factors and media, not only leading to the degradation and injury of the cartilage tissue and promoting the development of osteoarthritis but also resulting in synovial fibrosis and joint stiffness. Therefore, the synovium has attracted increasing attention in the research of OA, and the study of the mechanism of activation of FLSs and the fibrosis of joint synovium may shed new light on OA treatment. By using high-throughput screening, we have identified that hsa-miR338-3p is significantly downregulated in the synovial tissue and joint effusion from OA patients. A functional study showed that overexpression of hsa-miR338-3p in the FLSs inhibited the TGF-β1-induced overactivation of the TGF-β/Smad fibrosis regulation pathway by suppressing TRAP-1 expression and thus reducing the TGF-β1-induced activation of the FLSs and the expression of vimentin and collagen I, two fibrosis markers. Meanwhile, a mechanism study also showed that the upregulation of hsa-miR338-3p reduced Smad2/3 phosphorylation by suppressing TRAP-1 and thus inhibited the TGF-β/Smad pathway and TIMP1, a downstream protein. The present study, for the first time, illustrates the role of hsa-miR338-3p in synovial fibrosis in OA patients and the related mechanism, which is of importance to the treatment of OA and its complications by targeting the FLSs and synovial tissue. Hsa-miR338-3p not only has the potential to be a target for the gene therapy of OA but also has the potential to be a new marker for the diagnosis of clinical progression in OA patients.

Topics: Adult; Aged; Down-Regulation; Female; Fibroblasts; Fibrosis; HSP90 Heat-Shock Proteins; Humans; Male; MicroRNAs; Middle Aged; Osteoarthritis; Synovial Membrane; Synoviocytes; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta

Transforming growth factor-β (TGFβ) is a major regulator of cartilage homeostasis and its deregulation has been associated with osteoarthritis (OA). Deregulation of the TGFβ pathway in mesenchymal stem cells (MSCs) has been proposed to be at the onset of OA. Using a secretome analysis, we identified a member of the TGFβ family, TGFβ-induced protein (TGFβi or βIGH3), expressed in MSCs and we investigated its function and regulation during OA.. Cartilage, bone, synovium, infrapatellar fat pad and bone marrow-MSCs were isolated from patients with OA or healthy subjects. Chondrogenesis of BM-MSCs was induced by TGFβ3 in micropellet culture. Expression of TGFβi was quantified by RT-qPCR, ELISA or immunohistochemistry. Role of TGFβi was investigated in gain and loss of function experiments in BM-MSCs and chondrocytes.. TGFβi was up-regulated in early stages of chondrogenesis and its knock-down in BM-MSCs resulted in the down-regulation of mature and hypertrophic chondrocyte markers. It likely occurred through the modulation of adhesion molecules including integrin (ITG)β1, ITGβ5 and N-cadherin. We also showed that TGFβi was upregulated in vitro in a model of OA chondrocytes, and its silencing enhanced the hypertrophic marker type X collagen. In addition, TGFβi was up-regulated in bone and cartilage from OA patients while its expression was reduced in BM-MSCs. Similar findings were observed in a murine model of OA.. Our results revealed a dual role of TGFβi during chondrogenesis and pointed its deregulation in OA joint tissues. Modulating TGFβi in BM-MSCs might be of interest in cartilage regenerative medicine.

Topics: Animals; Chondrocytes; Chondrogenesis; Humans; Mesenchymal Stem Cells; Mice; Middle Aged; Osteoarthritis; Transforming Growth Factor beta

Prg4, also known as Lubricin, acts as a joint/boundary lubricant. Prg4 has been used to prevent surgically induced osteoarthritis (OA) in mice. Surgically induced OA serves as a good model for post-traumatic OA but is not ideal for recapitulating age-related OA. Reduced expression of the TGF-β type II receptor (TGFβR2) is associated with age-related OA in clinical samples, so we previously characterized a mouse model that exhibits OA due to expression of a mutated dominant-negative form of TGFβR2 (DNIIR). Prg4 expression was significantly reduced in DNIIR mice. Furthermore, we showed that Prg4 was a transcriptional target of TGF-ß via activation of Smad3, the main signal transducing protein for TGF-ß. The objective of the present study was to determine whether maintenance of Prg4, a down-stream transcriptional target of TGF-ß, prevents OA associated with attenuated TGF-ß signaling in mice.. Wild-type, DNIIR, and bitransgenic mice that express both DNIIR and Prg4, were compared. Mice were assessed with a foot misplacement behavioral test, μCT, histology, and Western blot.. Compared to DNIIR mice, bitransgenic DNIIR+Prg4 mice missed 1.3 (0.4, 2.1) fewer steps while walking (mean difference (95% confidence interval)), exhibited a cartilage fibrillation score that was 1.8 (0.4, 3.1) points lower, exhibited cartilage that was 28.2 (0.5, 55.9) μm thicker, and exhibited an OARSI score that was 6.8 (-0.9, 14.5) points lower. However, maintenance of Prg4 expression did not restore levels of phosphorylated Smad3 in DNIIR mice, indicating Prg4 does not simply stimulate TGF-ß signaling.. Our results indicate that maintenance of Prg4 expression prevents OA progression associated with reduced TGF-β signaling in mice. Since there was no evidence that Prg4 acts by stimulating the TGF-ß signaling cascade, we propose that Prg4, a transcriptional target of TGF-ß, attenuates OA progression through its joint lubrication function.

Topics: Animals; Cartilage, Articular; Disease Models, Animal; Disease Progression; Down-Regulation; Female; Male; Mice; Mice, Inbred C57BL; Osteoarthritis; Protective Factors; Proteoglycans; Receptor, Transforming Growth Factor-beta Type II; Signal Transduction; Transforming Growth Factor beta

Osteoarthritis (OA) is a degenerative condition caused by dysregulation of multiple molecular signalling pathways. Such dysregulation results in damage to cartilage, a smooth and protective tissue that enables low friction articulation of synovial joints. Matrix metalloproteinases (MMPs), especially MMP-13, are key enzymes in the cleavage of type II collagen which is a vital component for cartilage integrity. Transforming growth factor beta (TGFβ) can protect against pro-inflammatory cytokine-mediated MMP expression. With age there is a change in the ratio of two TGFβ type I receptors (Alk1/Alk5), a shift that results in TGFβ losing its protective role in cartilage homeostasis. Instead, TGFβ promotes cartilage degradation which correlates with the spontaneous development of OA in murine models. However, the mechanism by which TGFβ protects against pro-inflammatory responses and how this changes with age has not been extensively studied. As TGFβ signalling is complex, we used systems biology to combine experimental and computational outputs to examine how the system changes with age. Experiments showed that the repressive effect of TGFβ on chondrocytes treated with a pro-inflammatory stimulus required Alk5. Computational modelling revealed two independent mechanisms were needed to explain the crosstalk between TGFβ and pro-inflammatory signalling pathways. A novel meta-analysis of microarray data from OA patient tissue was used to create a Cytoscape network representative of human OA and revealed the importance of inflammation. Combining the modelled genes with the microarray network provided a global overview into the crosstalk between the different signalling pathways involved in OA development. Our results provide further insights into the mechanisms that cause TGFβ signalling to change from a protective to a detrimental pathway in cartilage with ageing. Moreover, such a systems biology approach may enable restoration of the protective role of TGFβ as a potential therapy to prevent age-related loss of cartilage and the development of OA.

Topics: Aging; Cell Line; Chondrocytes; Gene Expression Profiling; Humans; Osteoarthritis; Signal Transduction; Systems Biology; Transforming Growth Factor beta

CEMIP (for "Cell migration-inducing protein" also called KIAA1199 and Hybid for "Hyaluronan-binding protein") expression is increased in cancers and described as a regulator of cell survival, growth and invasion. In rheumatoid arthritis, CEMIP is referred to as an angiogenic marker and participates in hyaluronic acid degradation. In this study, CEMIP expression is investigated in healthy and osteoarthritis (OA) cartilage from human and mouse. Its role in OA physiopathology is deciphered, specifically in chondrocytes proliferation and dedifferentiation and in the extracellular matrix remodeling. To this end, CEMIP, αSMA and types I and III collagen expressions were assessed in human OA and non-OA cartilage. CEMIP expression was also investigated in a mouse OA model. CEMIP expression was studied in vitro using a chondrocyte dedifferentiation model. High-throughput RNA sequencing was performed on chondrocytes after CEMIP silencing. Results showed that CEMIP was overexpressed in human and murine OA cartilage and along chondrocytes dedifferentiation. Most of genes deregulated in CEMIP-depleted cells were involved in cartilage turnover (e.g., collagens), mesenchymal transition and fibrosis. CEMIP regulated β-catenin protein level. Moreover, CEMIP was essential for chondrocytes proliferation and promoted αSMA expression, a fibrosis marker, and TGFβ signaling towards the p-Smad2/3 (Alk5/PAI-1) pathway. Interestingly, CEMIP was induced by the pSmad1/5 (Alk1) pathway. αSMA and type III collagen expressions were overexpressed in human OA cartilage and along chondrocytes dedifferentiation. Finally, CEMIP was co-expressed in situ with αSMA in all OA cartilage layers. In conclusion, CEMIP was sharply overexpressed in human and mouse OA cartilage and along chondrocytes dedifferentiation. CEMIP-regulated transdifferentiation of chondrocytes into "chondro-myo-fibroblasts" expressing α-SMA and type III collagen, two fibrosis markers. Moreover, these "chondro-myo-fibroblasts" were found in OA cartilage but not in healthy cartilage.

Topics: Actins; Adult; Aged; Aged, 80 and over; Animals; beta Catenin; Cartilage, Articular; Cell Differentiation; Cell Proliferation; Cells, Cultured; Chondrocytes; Collagen Type I; Collagen Type III; Disease Models, Animal; Extracellular Matrix; Female; Fibrosis; High-Throughput Nucleotide Sequencing; Humans; Hyaluronoglucosaminidase; Male; Mice; Mice, Inbred C57BL; Middle Aged; Osteoarthritis; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta

Osteoarthritis (OA) is a conversant joint disease, which seriously threatens the health of the elderly, and even leads to disability. Long non-coding RNA-activated by transforming growth factor beta (lncRNA-ATB) has been reported in diverse cancers. However, the functions of lncRNA-ATB in OA remain uninvestigated. The current study aimed to explore the impacts of lncRNA-ATB on lipopolysaccharide (LPS)-induced inflammatory injury in ATDC5 cells and to uncover the underlying mechanism. LPS-induced ATDC5 cell injury model was constructed, and the effects of lncRNA-ATB on LPS-injured cells were explored via analyzing cell viability, apoptosis, iNOS, COX-2, and inflammatory cytokines (IL-6 and TNF-α). Subsequently, the relationship between lncRNA-ATB and microRNA (miR)-223 was detected, and whether miR-223 was involved in modulating LPS-induced cells injury in ATDC5 cells was investigated. Finally, MyD88/NF-κB and p38MAPK pathways were assessed to explore the underlying mechanism. Results showed that LPS repressed cell viability, induced apoptosis, and promoted iNOS, COX-2, IL-6 and TNF-α expression. Additionally, we observed that lncRNA-ATB expression was down-regulated in LPS-injured cells, and lncRNA-ATB overexpression significantly alleviated LPS-induced inflammatory injury in ATDC5 cells. Interesting results revealed that miR-223 expression was down-regulated by lncRNA-ATB and miR-223 overexpression declined the protective effect of lncRNA-ATB on LPS-injured ATDC5 cells. Further, the signaling pathway experiments showed that lncRNA-ATB inhibited MyD88/NF-κB and p38MAPK pathways by down-regulating miR-223 in LPS-injured cells. These data demonstrated that lncRNA-ATB protected ATDC5 cells against LPS-induced inflammatory injury by repressing MyD88/NF-κB and p38MAPK pathways, which was mediated by down-regulation of miR-223.

Topics: Aged; Animals; Anti-Inflammatory Agents; Apoptosis; Cell Line; Chondrocytes; Cyclooxygenase 2; Disease Models, Animal; Humans; Inflammation; Interleukin-6; Lipopolysaccharides; Mice; MicroRNAs; Nitric Oxide Synthase Type II; Osteoarthritis; RNA, Long Noncoding; Signal Transduction; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Synovial fibrosis is a pathological process that is observed in several musculoskeletal disorders and characterized by the excessive deposition of extracellular matrix, as well as cell migration and proliferation. Despite the fact that glucocorticoids are widely employed in the treatment of rheumatic pathologies such as osteoarthritis (OA) and rheumatoid arthritis, the mechanisms by which glucocorticoids act in the joint and their impacts on pro-fibrotic pathways are still unclear.. Human OA synovial fibroblasts were obtained from knee and hip joints. Cells were treated with prednisolone (1 mM) or transforming growth factor-beta 1 (TGF-β1) (10 ng/ml) for 1 and 7 days for quantification of RNA and protein expression (by real-time quantitative reverse transcription-PCR and western blot, respectively), 72 h for immunocytochemistry analysis, and 48 h for proliferation (by BrdU assay) and migration (by wound assay) studies. In addition, cells were preincubated with prednisolone and/or the peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist 15-deoxy-Δ-12,14-prostaglandin J2 (15d-PGJ2) for 6 h before adding TGF-β1. pSmad1/5, pSmad2 and β-catenin levels were analyzed by Western blot. The activin receptor-like kinase-5 (ALK-5) inhibitor (SB-431542) was employed for the mechanistic assays.. Prednisolone showed a predominant anti-fibrotic impact on fibroblast-like synoviocytes as it attenuated the spontaneous and TGF-β-induced gene expression of pro-fibrotic markers. Prednisolone also reduced α-sma protein and type III collagen levels, as well as cell proliferation and migration after TGF-β stimulation. However, prednisolone did not downregulate the gene expression of all the pro-fibrotic markers tested and did not restore the reduced PPAR-γ levels after TGF-β stimulation. Interestingly, anti-fibrotic actions of the glucocorticoid were reinforced in the presence of the PPAR-γ agonist 15d-PGJ2. Combined pretreatment modulated Smad2/3 levels and, similar to the ALK-5 inhibitor, blocked β-catenin accumulation elicited by TGF-β.. Prednisolone, along with 15d-PGJ2, modulates pro-fibrotic pathways activated by TGF-β in synovial fibroblasts at least partially through the inhibition of ALK5/Smad2 signaling and subsequent β-catenin accumulation. These findings shed light on the potential therapeutic effects of glucocorticoids treatment combined with a PPAR-γ agonist against synovial fibrosis, although future studies are warranted to further evaluate this concern.

Topics: Adult; Aged; beta Catenin; Cells, Cultured; Female; Fibroblasts; Fibrosis; Humans; Male; Middle Aged; Osteoarthritis; PPAR gamma; Prednisolone; Prostaglandin D2; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Topics: Chondrocytes; Humans; Osteoarthritis; Transforming Growth Factor beta

Epidemiological investigations indicate that effects related to prenatal adverse environments on the organs of the offspring could continue to adulthood. This study intends to confirm that prenatal nicotine exposure (PNE) increases the susceptibility of osteoarthritis (OA) in the male offspring, and to explore the potential intrauterine programming mechanism. During pregnancy, rats were divided into a PNE group and a control group. After birth, rats were given a high-fat diet for 6 months and long-distance running for 6 weeks. The rats were euthanized at 18 months after birth (PM18) and on gestational day 20 (GD20), respectively. Knee joints were collected for histochemistry, immunohistochemistry, and quantitative polymerase chain reaction (qPCR) assays. Histological analyses and the Mankin's score showed increased cartilage destruction and accelerated OA progression in adult offspring from the PNE group. Immunohistochemistry results showed decreased expression of transforming growth factor beta (TGFβ) signaling pathway. Furthermore, the expression of apoptosis factors (caspase-3 and caspase-8), inflammatory factors [interleukin (IL)-1, IL-6] and matrix degradation enzymes [matrix metalloproteinase (MMP)-3, MMP-13] were also significantly increased. Traced back to the intrauterine period, it was found that the number of chondrocytes and the contents of Col2A1 and aggrecan in the matrix in the PNE group were decreased. And, the expression of the TGFβ signaling pathway was inhibited. These results suggested that PNE enhanced the susceptibility of OA in male elderly offspring rats by down-regulating TGFβ signaling, which increased articular cartilage local inflammation, matrix degradation, and cell apoptosis. This study confirmed the developmental origin of OA, and clarified the congenital and the living environment impact on the occurrence and development of OA. Our findings provide a theoretical and experimental basis for OA early prevention.

Topics: Age Factors; Aggrecans; Animals; Apoptosis; Caspase 3; Caspase 8; Chondrocytes; Chondrogenesis; Collagen Type II; Female; Gestational Age; Interleukin-1; Interleukin-6; Joints; Male; Maternal Exposure; Matrix Metalloproteinase 13; Matrix Metalloproteinase 3; Nicotine; Nicotinic Agonists; Osteoarthritis; Pregnancy; Prenatal Exposure Delayed Effects; Rats, Wistar; Risk Factors; Sex Factors; Signal Transduction; Transforming Growth Factor beta

Osteoarthritis (OA) is a common degenerative joint disease; however, its underlying pathogenesis remains to be elucidated. Previous studies have demonstrated that the transforming growth factor‑β (TGF‑β) signaling pathway has a role in the initiation and development of OA. Additionally, latent TGF‑β‑binding protein‑1 (LTBP‑1) modulates the activity of the TGF‑β‑mothers against decapentaplegic (Smad) signaling pathway in numerous diseases, including malignant glioma. The present study demonstrated that expression of LTBP‑1 is increased in OA synovial tissues compared with normal synovial tissues. The effect of TGF‑β was identified to be mediated by phosphorylated(p)‑(Smad)2/3, which may activate activin‑like kinase (ALK)5 receptor, and by p‑Smad1/5/8, which may induce ALK1, thereby stimulating expression of matrix metalloproteinase‑(MMP)‑13 in OA fibroblast‑like synoviocytes (FLS). Compared with normal FLS, OA FLS demonstrated an increased p‑Smad1/5/8:p‑Smad2 ratio, which led to elevated MMP‑13 expression and aggravation of OA. Furthermore, knockdown of the LTBP‑1 gene by siRNA transfection in OA FLS reduced p‑Smad1/5/8 expression without affecting TGF‑β mRNA levels, although p‑Smad2 expression increased. It was also demonstrated that OA FLS exhibited increased proliferation compared with normal FLS in vitro. Furthermore, siRNA‑mediated downregulation of LTBP‑1 reduced proliferation of OA FLS. In conclusion, the present study demonstrated that an alteration in the p‑Smad1/5/8:p‑Smad2 ratio as well as association between p‑Smad1/5/8 and MMP‑13 expression in human OA FLS, may contribute to the development of OA. The results of the present study suggested that LTBP‑1 is a modulator of the TGF‑β signaling pathway in human OA FLS, which may aid in elucidating the mechanism underlying the pathology of OA.

Topics: Aged; Cells, Cultured; Female; Fibroblasts; Humans; Latent TGF-beta Binding Proteins; Male; Middle Aged; Osteoarthritis; Synovial Membrane; Synoviocytes; Transforming Growth Factor beta

Aging is a main risk factor for osteoarthritis (OA). FoxO transcription factors protect against cellular and organismal aging, and FoxO expression in cartilage is reduced with aging and in OA. To investigate the role of FoxO in cartilage, Col2Cre-FoxO1, 3, and 4 single knockout (KO) and triple KO mice (Col2Cre-TKO) were analyzed. Articular cartilage in Col2Cre-TKO and Col2Cre-FoxO1 KO mice was thicker than in control mice at 1 or 2 months of age. This was associated with increased proliferation of chondrocytes of Col2Cre-TKO mice in vivo and in vitro. OA-like changes developed in cartilage, synovium, and subchondral bone between 4 and 6 months of age in Col2Cre-TKO and Col2Cre-FoxO1 KO mice. Col2Cre-FoxO3 and FoxO4 KO mice showed no cartilage abnormalities until 18 months of age when Col2Cre-FoxO3 KO mice had more severe OA than control mice. Autophagy and antioxidant defense genes were reduced in Col2Cre-TKO mice. Deletion of FoxO1/3/4 in mature mice using Aggrecan(Acan)-CreERT2 (AcanCreERT-TKO) also led to spontaneous cartilage degradation and increased OA severity in a surgical model or treadmill running. The superficial zone of knee articular cartilage of Col2Cre-TKO and AcanCreERT-TKO mice exhibited reduced cell density and markedly decreased

Topics: Animals; Animals, Newborn; Autophagy; Body Size; Bone and Bones; Cartilage, Articular; Cell Proliferation; Cell Survival; Chondrocytes; Forkhead Transcription Factors; Gene Deletion; Gene Expression Regulation; Growth Plate; Homeostasis; Humans; Interleukin-1beta; Mice, Knockout; Osteoarthritis; Proteoglycans; Transforming Growth Factor beta

Articular cartilage degeneration plays a key role in the pathogenesis of osteoarthritis (OA). Bushenhuoxue formula (BSHXF) has been widely used in the treatment of OA in clinics. However, the molecular mechanisms responsible for the chondroprotective effect of BSHXF remain to be elucidated. The purpose of this study was to explore the effects of BSHXF on OA mice model.. In this study, we investigated the effects of BSHXF on destabilization of the medial meniscus (DMM)-induced chondrocyte degradation in OA mice model. At 12 weeks post-surgery, the joints were harvested for tissue analyses, including histology, histomorphometry, TUNEL, OARSI scoring, micro-CT and immunohistochemistry for COL2, TGFBR2, pSMAD2 and MMP13. Additionally, we also evaluated the effects of BSHXF on Mmp13 mRNA and protein expression in chondrogenic ATDC5 cells through real-time PCR and Western blot respectively. Moreover, we investigated the chondroprotective effect of BSHXF on mice with Tgfbr2 conditional knockout (Tgfbr2. Amelioration of cartilage degradation and chondrocyte apoptosis were observed in DMM-induced mice, with increases in cartilage area and thickness, proteoglycan matrix, COL2 content and decreases in OARSI score at 12 weeks post surgery. Moreover, the elevated TGFBR2 and pSMAD2, and reduced MMP13 positive cells were also revealed in DMM-induced mice treated with BSHXF. Besides, decreased Mmp13 mRNA and protein expression were observed inchondrogenic ATDC5 cells culture in serum containing BSHXF. As expected, Tgfbr2. BSHXF could inhibit cartilage degradation through TGF-β/MMP13 signaling, and be considered a good option for the treatment of OA.

Topics: Animals; Cartilage, Articular; Chondrocytes; Disease Models, Animal; Disease Progression; Drugs, Chinese Herbal; Matrix Metalloproteinase 13; Menisci, Tibial; Mice, Inbred C57BL; Osteoarthritis; Phenotype; Phosphorylation; Rats, Sprague-Dawley; Receptor, Transforming Growth Factor-beta Type II; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta; Up-Regulation

Based on our previous findings that prenatal ethanol exposure in offspring increased susceptibility to adult osteoarthritis, this study aimed to further investigate the direct toxicity of ethanol on fetal articular cartilage development. Rat bone marrow-derived stroma cells were capsulated in alginate beads, incubated in a chondrogenic differentiation medium, and cultured for 4 weeks with ethanol treatment at concentrations of 0, 4, 20, and 100 mM. Pregnant rats were treated with ethanol (4 g/kg/day) from gestational days (GDs) 9 to 20. At GD20 and postnatal weeks 2, 6, and 12, 8 male offspring were sacrificed, and 8 male offspring rats of 8-weeks old in each group were treated with or without intraarticular injection of papain for 4 weeks to verify the susceptibility of adult osteoarthritis. Ethanol treatment resulted in poor differentiation of bone marrow-derived stroma cells to chondrocytes and suppressed the expression of the transforming growth factor-β (TGFβ)-smad2/3-Sox9 signaling pathway. In animal experiments, the shape of articular cartilage in the ethanol treatment group was more disordered than that of the control group, the matrix was not deep, and the cartilage was thin, which showed poor cartilage development. The TGFβ signaling pathway in the ethanol treatment group was persistently low at all time points. After intraarticular injection of papain, histological analyses, and the Mankin score revealed increased cartilage destruction in the ethanol treatment group. Ethanol caused articular cartilage dysplasia that was programmed in adulthood via a low-functional TGFβ signaling pathway, and the tolerance of this articular cartilage to external stimuli was significantly decreased.

Topics: Animals; Cartilage, Articular; Ethanol; Female; Fetal Alcohol Spectrum Disorders; Fetal Development; Male; Maternal Exposure; Osteoarthritis; Pregnancy; Prenatal Exposure Delayed Effects; Rats; Transforming Growth Factor beta

The success of cell-based approaches for the treatment of cartilage defects requires an optimal autologous cell source with chondrogenic differentiation ability that maintains its differentiated properties and stability following implantation. The objective of this study was to compare the chondrogenic capacity of mesenchymal stem cells (MSCs) isolated from lipoaspirates (ASCs) and the infrapatellar fat pad (IFPSCs) of osteoarthritic patients and treated with transforming growth factor (TGF)-β family-related growth factors. Cells were cultured for 6 weeks in a 3D pellet culture system with the chimeric activin A/bone morphogenic protein (BMP)-2 ligand (AB235), the chimeric nodal/BMP-2 ligand (NB260) or BMP-2. To investigate the stability of the new cartilage, ASCs-treated pellets were transplanted subcutaneously into severe combined immunodeficiency (SCID) mice. Histological and immunohistochemical assessment confirmed that the growth factors induced cartilage differentiation in both isolated cell types. However, reverse transcription-quantitative PCR results showed that ASCs presented a higher chondrogenic potential than IFPSCs. In vivo results revealed that AB235-treated ASCs pellets were larger in size and could form stable cartilage-like tissue as compared to NB260-treated pellets, while BMP-2-treated pellets underwent calcification. The chondrogenic induction of ASCs by AB235 treatment was mediated by SMAD2/3 activation, as proved by immunofluorescence analysis. The results of this study indicated that the combination of ASCs and AB235 might lead to a cell-based cartilage regeneration treatment.

Topics: Adipose Tissue; Aged; Animals; Cell Differentiation; Cell Separation; Chondrogenesis; Female; Humans; Lipectomy; Male; Mice, SCID; Middle Aged; Osteoarthritis; Phenotype; Smad Proteins; Stem Cell Transplantation; Stem Cells; Transforming Growth Factor beta

The aim of this study was to compare the effects of photobiomodulation (PBM) associated with an aerobic and an aquatic exercise training on the degenerative process related to osteoarthritis (OA) in the articular cartilage in rats. Fifty male Wistar rats were randomly divided into 5 groups: OA control group (CG), OA plus aerobic training group (AET), OA plus aquatic training group (AQT), OA plus aerobic training associated with PBM group (AETL), OA plus aquatic training associated with PBM group (AQTL). The aerobic training (treadmill; 16 m/min; 50 min/day) and the aquatic training (water jumping; 50-80% of their body mass) started 4 weeks after the surgery and they were performed 3 days/week for 8 weeks. Moreover, PBM was performed after the physical exercise trainings on the left joint. Morphological characteristics and immunoexpression of IL-10, TGF-β, and collagen type I (Col I) and II (Col II) of the articular cartilage were evaluated. The results showed that all the treated groups (exercise and PBM) presented less intense signs of degradation (measured by histopathological analysis and OARSI grade system). Additionally, aerobic and aquatic exercise training rats (associated or not with PBM) showed increased IL-10 (AET p = 0.0452; AETL p = 0.03; AQTL p = 0.0193) and Col II (AET p = 0.012; AQT p = 0.0437; AETL p = 0.0001; AQTL p = 0.0001) protein expression compared to CG. Furthermore, a statistically higher TGF-β expression was observed in AET (p = 0.0084) and AETL (p = 0.0076) compared to CG. These results suggest that PBM associated with aerobic and aquatic exercise training were effective in mediating chondroprotective effects and maintaining the integrity of the articular tissue in the knees of OA rats.

Topics: Animals; Cartilage, Articular; Collagen Type I; Collagen Type II; Disease Models, Animal; Humans; Interleukin-10; Low-Level Light Therapy; Male; Osteoarthritis; Physical Conditioning, Animal; Rats, Wistar; Transforming Growth Factor beta

Osteoarthritis (OA) is characterized by synovitis and synovial fibrosis. Synoviocytes are fibroblast-like resident cells of the synovium that are activated by transforming growth factor (TGF)-β to proliferate, migrate, and produce extracellular matrix. Synoviocytes secrete hyaluronan (HA) and proteoglycan-4 (PRG4). HA reduces synovial fibrosis in vivo, and the Prg4

Topics: Actins; Aged; Animals; Colforsin; Collagen Type I; Collagen Type I, alpha 1 Chain; Cyclic AMP; Extracellular Matrix; Female; Fibroblasts; Fibrosis; Humans; Hyaluronic Acid; Male; Mice; Middle Aged; Osteoarthritis; Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase; Proteoglycans; Synovial Membrane; Synoviocytes; Transforming Growth Factor beta

One mechanism by which cartilage responds to mechanical load is by releasing heparin-bound growth factors from the pericellular matrix (PCM). By proteomic analysis of the PCM, we identified connective tissue growth factor (CTGF) and here investigate its function and mechanism of action.. Recombinant CTGF (rCTGF) was used to stimulate human chondrocytes for microarray analysis. Endogenous CTGF was investigated by in vitro binding assays and confocal microscopy. Its release from cut cartilage (injury CM) was analysed by Western blot under reducing and non-reducing conditions. A postnatal, conditional Ctgf. The biological responses of rCTGF were TGFβ dependent. CTGF displaced latent TGFβ from cartilage and both were released on cartilage injury. CTGF and latent TGFβ migrated as a single high molecular weight band under non-reducing conditions, suggesting that they were in a covalent (disulfide) complex. This was confirmed by immunoprecipitation. Using Ctgf. CTGF is a latent TGFβ binding protein that controls the matrix sequestration and activation of TGFβ in cartilage. Deletion of CTGF in vivo caused a paradoxical increase in Smad2 phosphorylation resulting in thicker cartilage that was protected from OA.

Topics: Animals; Arthritis, Experimental; Cartilage, Articular; Cells, Cultured; Chondrocytes; Connective Tissue Growth Factor; Homeostasis; Humans; Mice, Knockout; Osteoarthritis; Proteoglycans; Proteomics; Receptors, Transforming Growth Factor beta; Recombinant Proteins; Smad2 Protein; Tissue Culture Techniques; Transforming Growth Factor beta

Topics: Cartilage, Articular; Connective Tissue Growth Factor; Homeostasis; Humans; Osteoarthritis; Transforming Growth Factor beta

Cartilage loss in osteoarthritis (OA) results from altered local production of growth factors and metalloproteases (MMPs). Furin, an enzyme involved in the protein maturation of MMPs, might regulate chondrocyte function. Here, we tested the effect of furin on chondrocyte catabolism and the development of OA. In primary chondrocytes, furin reduced the expression of MMP-13, which was reversed by treatment with the furin inhibitor α1-PDX. Furin also promoted the activation of Smad3 signaling, whereas activin receptor-like kinase 5 (ALK5) knockdown mitigated the effects of furin on MMP-13 expression. Mice underwent destabilization of the medial meniscus (DMM) to induce OA, then received furin (1 U/mice), α1-PDX (14 µg/mice) or vehicle. In mice with DMM, the OA score was lower with furin than vehicle treatment (6.42 ± 0.75 vs 9.16 ± 0.6, p < 0.01), and the number of MMP-13(+) chondrocytes was lower (4.96 ± 0.60% vs 20.96 ± 8.49%, p < 0.05). Moreover, furin prevented the increase in ALK1/ALK5 ratio in cartilage induced by OA. Conversely, α1-PDX had no effect on OA cartilage structure. These results support a protective role for furin in OA by maintaining ALK5 receptor levels and reducing MMP-13 expression. Therefore, furin might be a potential target mediating the development of OA.

Topics: Activin Receptors, Type I; Activin Receptors, Type II; Animals; Chondrocytes; Furin; Matrix Metalloproteinase 13; Mice; Osteoarthritis; Proprotein Convertases; Receptor, Transforming Growth Factor-beta Type I; Transforming Growth Factor beta

Osteoarthritis (OA) is a joint disease characterized by progressive degeneration of articular cartilage. Some features of OA, including chondrocyte hypertrophy and focal calcification of articular cartilage, resemble the endochondral ossification processes. Alterations in transforming growth factor β (TGFβ) signaling have been associated with OA as well as with chondrocyte hypertrophy. Our aim was to identify novel candidate genes implicated in chondrocyte hypertrophy during OA pathogenesis by determining which TGFβ-related genes are regulated during murine OA and endochondral ossification.. A list of 580 TGFβ-related genes, including TGFβ signaling pathway components and TGFβ-target genes, was generated. Regulation of these TGFβ-related genes was assessed in a microarray of murine OA cartilage: 1, 2 and 6 weeks after destabilization of the medial meniscus (DMM). Subsequently, genes regulated in the DMM model were studied in two independent murine microarray datasets on endochondral ossification: the growth plate and transient embryonic cartilage (joint development).. A total of 106 TGFβ-related genes were differentially expressed in articular cartilage of DMM-operated mice compared to sham-control. From these genes, 43 were similarly regulated during chondrocyte hypertrophy in the growth plate or embryonic joint development. Among these 43 genes, 18 genes have already been associated with OA. The remaining 25 genes were considered as novel candidate genes involved in OA pathogenesis and endochondral ossification. In supplementary data of published human OA microarrays we found indications that 15 of the 25 novel genes are indeed regulated in articular cartilage of human OA patients.. By focusing on TGFβ-related genes during OA and chondrocyte hypertrophy in mice, we identified 18 known and 25 new candidate genes potentially implicated in phenotypical changes in chondrocytes leading to OA. We propose that 15 of these candidates warrant further investigation as therapeutic target for OA as they are also regulated in articular cartilage of OA patients.

Topics: Animals; Cell Line; Chondrocytes; Databases, Genetic; Disease Models, Animal; Gene Expression Regulation; Hypertrophy; Joints; Male; Mice, Inbred C57BL; Oligonucleotide Array Sequence Analysis; Osteoarthritis; Reproducibility of Results; Transforming Growth Factor beta

Topics: Animals; Cartilage, Articular; Chondrocytes; Collagen Type II; Gene Expression Regulation, Developmental; Humans; Immunohistochemistry; Meniscus; Mice; Mice, Knockout; Osteoarthritis; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

The degradation of cruciate ligaments is frequently observed in degenerative joint diseases, such as osteo-arthritis (OA). The present study aimed to identify the differentially expressed microRNAs (miRNAs or miRs) in knee anterior cruciate ligament (ACL) tissues derived from patients with OA and in health subjects (non-OA). By using Affymetrix miRNA 4.0 microarrays, a total of 22 miRNAs (including let-7f-5p, miR-26b-5p and miR-146a-5p) were found to be upregulated, while 17 (including miR-18a-3p, miR-138-5p and miR-485-3p) were downregulated in the osteoarthritic ACL tissues (fold change ≥2, P-value <0.05). The expression levels of 12 miRNAs were validated by quantitative PCR, and the corresponding results revealed an excellent correlation with the microarray data (R2=0.889). Genes (such as a disintegrin and metalloproteinase domain with thrombospondin type-1 motifs, bone morphogenetic protein-2, runt related transcription factor-2, collagen-1A1 and 2, interleukin-6 and transforming growth factor-β) involved in cartilage development and remodeling, collagen biosynthesis and degradation, inflammatory response and extracellular matrix homeostasis were predicted as potential targets of the dysregulated miRNAs. Moreover, a large set of putative genes were enriched in OA pathogenesis‑associated pathways (such as mitogen-activated protein kinase and vascular endothelial growth factor signaling pathway). Collectively, the data from our study provides novel insight into the ligament injury-related miRNA dysregulation in patients with OA.

Topics: Aged; Anterior Cruciate Ligament; Bone Morphogenetic Protein 2; Case-Control Studies; Collagen Type I; Collagen Type I, alpha 1 Chain; Core Binding Factor Alpha 1 Subunit; Female; Fertilins; Gene Expression Profiling; Gene Expression Regulation; Gene Ontology; Humans; Interleukin-6; Knee Joint; Male; MicroRNAs; Middle Aged; Molecular Sequence Annotation; Oligonucleotide Array Sequence Analysis; Osteoarthritis; Real-Time Polymerase Chain Reaction; Transforming Growth Factor beta

Lymphocyte activation gene-3 (LAG-3) is a CD4 homologue expressed on the surface of activated conventional T cells and regulatory T (Treg) cells. In conventional T cells, LAG-3 acts as an inhibitory receptor of T cell inflammation. However, the role of LAG-3 in Treg cells remains controversial. In this study, we investigated the effect of LAG-3 on Tregs in osteoarthritis (OA). We observed that the proportion of LAG-3-expressing cells in CD4

Topics: Antigens, CD; Cell Differentiation; Forkhead Transcription Factors; Humans; Interleukin-10; Lymphocyte Activation; Lymphocyte Activation Gene 3 Protein; Osteoarthritis; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

The present study aimed to investigate the effect of tanshinone IIA on the degradation of articular cartilage in a rat model of osteoarthritis (OA). The OA rat model was established by anterior cruciate ligament transection (ACLT) and medial meniscus resection (MMx). The animals were treated for 28 days with 0.25‑0.5 mg/kg doses of tanshinone IIA following ACLT + MMx. The knee joints of the rats in the ACLT + MMx group exhibited marked alterations in articular cartilage histopathology and higher Mankin scores, compared with those in the normal group. Tanshinone IIA treatment at a dose of 0.5 mg/kg significantly inhibited cartilage degradation and improved Mankin scores in the OA rat model (P<0.002). Tanshinone IIA treatment completely inhibited the ACLT + MMx‑induced accumulation of inflammatory cells and disintegration of synovial lining in the rats. An increase in the dose of tanshinone IIA between 0.25 and 0.5 mg/kg reduced the proportion of apoptotic chrondrocytes from 41 to 2% on day 29. Treatment of the rats in the ACLT + MMx group with 0.5 mg/kg doses of tanshinone IIA markedly inhibited the expression level of matrix metalloproteinase and increased the expression of tissue inhibitor of metalloproteinase in the rat articular cartilage tissues. Tanshinone IIA treatment significantly reduced the levels of inflammatory cytokines, including interleukin‑1β, tumor necrosis factor‑α and nitric oxide in rat serum samples. The protein expression levels of bone morphogenetic protein and transforming growth factor‑β were significantly increased by tanshinone IIA in the ACLT + MMx rats. Therefore, tanshinone IIA inhibited articular cartilage degradation through inhibition of apoptosis and expression levels of inflammatory cytokines, offering potential for use in the treatment of OA.

Topics: Abietanes; Animals; Anterior Cruciate Ligament; Apoptosis; Bone Morphogenetic Proteins; Cartilage, Articular; Chondrocytes; Cytokines; Disease Models, Animal; Inflammation; Interleukin-1beta; Male; Menisci, Tibial; Nitric Oxide; Osteoarthritis; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Recombinant adeno-associated virus (rAAV) vectors are clinically adapted vectors to durably treat human osteoarthritis (OA). Controlled delivery of rAAV vectors via polymeric micelles was reported to enhance the temporal and spatial presentation of the vectors into their targets. Here, we tested the feasibility of delivering rAAV vectors via poly (ethylene oxide) (PEO) and poly (propylene oxide) (PPO) (poloxamer and poloxamine) polymeric micelles as a means to overexpress the therapeutic factor transforming growth factor-beta (TGF-β) in human OA chondrocytes and in experimental human osteochondral defects. Application of rAAV-human transforming growth factor-beta using such micelles increased the levels of TGF-β transgene expression compared with free vector treatment. Overexpression of TGF-β with these systems resulted in higher proteoglycan deposition and increased cell numbers in OA chondrocytes. In osteochondral defect cultures, a higher deposition of type-II collagen and reduced hypertrophic events were noted. Delivery of therapeutic rAAV vectors via PEO-PPO-PEO micelles may provide potential tools to remodel human OA cartilage.

Topics: Cartilage, Articular; Cell Proliferation; Chondrocytes; Dependovirus; Gene Transfer Techniques; Genetic Vectors; Humans; Hypertrophy; Micelles; Models, Biological; Osteoarthritis; Polyethylene Glycols; Propylene Glycols; Transforming Growth Factor beta; Transgenes

OBJECTIVE To determine effects of transforming growth factor (TGF)-β and interleukin (IL)-1β on inflammatory markers in cultured canine chondrocytes to clarify the role of these cytokines in osteoarthritis of dogs. SAMPLE Pooled chondrocytes isolated from the stifle joints of 4 adult dogs. PROCEDURES Chondrocytes were isolated, cultured, and frozen at -80°C. Pooled cells were incubated in medium with or without TGF-β (1 or 10 ng/mL) and subsequently stimulated with IL-1β (10 ng/mL). Concentrations of nitric oxide (NO) and prostaglandin (PG) E were measured in culture supernatants. Gene expression of matrix metalloproteinase (MMP)-3, tissue inhibitor of metalloproteinase (TIMP)-2, inducible NO synthase (iNOS), and cyclooxygenase (COX)-2 was quantified by use of real-time quantitative PCR assays. RESULTS Stimulation with IL-1β increased gene expression of all inflammatory markers, except for TIMP-2. Incubation with TGF-β resulted in a significant decrease in MMP-3 and TIMP-2 mRNA concentrations but had no effect on PGE and NO concentrations. For cells treated with TGF-β followed by IL-1β, concentrations of PGE and NO were lower, compared with concentrations for IL-1β control cells. Furthermore, IL-1β-induced gene expression of iNOS, MMP-3, and COX-2 was downregulated. However, the IL-1β-induced downregulation of TIMP-2 gene expression was partially restored by pretreatment with TGF-β. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that IL-1β increased the expression of inflammatory genes and mediators, and TGF-β largely attenuated the IL-1β-mediated inflammatory response. Therefore, TGF-β might be a novel target for use in the prevention and treatment of cartilage breakdown in dogs with osteoarthritis.

Topics: Animals; Cartilage; Cells, Cultured; Chondrocytes; Cyclooxygenase 2; Dog Diseases; Dogs; Gene Expression; Inflammation Mediators; Interleukin-1beta; Matrix Metalloproteinase 3; Nitric Oxide; Nitric Oxide Synthase Type II; Osteoarthritis; Tissue Inhibitor of Metalloproteinase-2; Transforming Growth Factor beta

The aim of this study was to show how computational models can be used to increase our understanding of the role of microRNAs in osteoarthritis (OA) using miR-140 as an example. Bioinformatics analysis and experimental results from the literature were used to create and calibrate models of gene regulatory networks in OA involving miR-140 along with key regulators such as NF-κB, SMAD3, and RUNX2. The individual models were created with the modelling standard, Systems Biology Markup Language, and integrated to examine the overall effect of miR-140 on cartilage homeostasis. Down-regulation of miR-140 may have either detrimental or protective effects for cartilage, indicating that the role of miR-140 is complex. Studies of individual networks in isolation may therefore lead to different conclusions. This indicated the need to combine the five chosen individual networks involving miR-140 into an integrated model. This model suggests that the overall effect of miR-140 is to change the response to an IL-1 stimulus from a prolonged increase in matrix degrading enzymes to a pulse-like response so that cartilage degradation is temporary. Our current model can easily be modified and extended as more experimental data become available about the role of miR-140 in OA. In addition, networks of other microRNAs that are important in OA could be incorporated. A fully integrated model could not only aid our understanding of the mechanisms of microRNAs in ageing cartilage but could also provide a useful tool to investigate the effect of potential interventions to prevent cartilage loss.

Topics: Computer Simulation; Humans; Interleukin-1; Matrix Metalloproteinases; MicroRNAs; Osteoarthritis; SOX9 Transcription Factor; Systems Biology; Transforming Growth Factor beta

Interterritorial regions of articular cartilage matrix are rich in decorin, a small leucine-rich proteoglycan and important structural protein, also involved in many signalling events. Decorin sequesters transforming growth factor β (TGFβ), thereby regulating its activity. Here, we analysed whether increased bioavailability of TGFβ in decorin-deficient (Dcn. Unchallenged knee cartilage was analysed by atomic force microscopy (AFM) and immunohistochemistry. Active transforming growth factor β-1 (TGFβ1) content within cultured chondrocyte supernatants was measured by ELISA. Quantitative real-time (RT)-PCR was used to analyse mRNA expression of glycosaminoglycan (GAG)-modifying enzymes in C28/I2 cells following TGFβ1 treatment. In addition, OA was induced in Dcn. AFM analysis revealed a strikingly higher compressive stiffness in Dcn. Our study demonstrates that the disruption of decorin-restricted TGFβ signalling leads to higher stiffness of articular cartilage matrix, rendering joints more resistant to OA. Therefore, the loss of an important structural component can improve cartilage homeostasis.

Topics: Animals; Arthritis, Experimental; Biomechanical Phenomena; Cartilage, Articular; Decorin; Enzyme-Linked Immunosorbent Assay; Glycosaminoglycans; Immunohistochemistry; Mice; Mice, Knockout; Microscopy, Atomic Force; Osteoarthritis; Physical Conditioning, Animal; Real-Time Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta

Low-density lipoproteins (LDL) in inflamed synovium is oxidized and taken-up by synoviocytes. In this study, we investigate whether direct injection of oxidized LDL (oxLDL) into a normal murine knee joint induces joint pathology and whether synovial macrophages are involved in that process.. Synovium was obtained from end-stage osteoarthritis (OA) patients in order to analyze LDL-uptake. Murine knee joints were injected five consecutive days with oxLDL, LDL, or vehicle (phosphate buffered saline (PBS)). This procedure was repeated in mice depleted of synovial macrophages by intra-articular injection of clodronate liposomes 7 days prior to the consecutive injections. Joint pathology was investigated by immunohistochemistry, flow cytometry (FCM) and synovial RNA expression and protein production.. Synovial tissue of OA patients showed extensive accumulation of apolipoprotein B. Multiple injections of oxLDL in murine knee joints significantly increased TGF-β activity in synovial wash-outs, but did not induce catabolic or inflammatory processes. In contrast, repeated injections of oxLDL in macrophage-depleted knee joints led to increased synovial thickening in combination with significantly upregulated protein and RNA levels of CCL2 and CCL3. FCM-analyses revealed increased presence of monocytes and neutrophils in the synovium, which was confirmed by immunohistochemistry. Also protein levels of S100A8/A9 were significantly increased in synovial wash-outs of oxLDL-injected joints, as was expression of aggrecanase-induced neo-epitopes. Interestingly, no raise in TGF-β concentrations was measured in macrophage-depleted joints.. OxLDL can affect joint pathology, since synovial macrophages promote anabolic processes after oxLDL injections. In absence of synovial macrophages, however, oxLDL induces production of pro-inflammatory mediators and aggrecanase activity combined with increased influx of monocytes and neutrophils.

Topics: Animals; Calgranulin A; Calgranulin B; Humans; Injections, Intra-Articular; Lipoproteins, LDL; Macrophages; Mice; Mice, Inbred C57BL; Osteoarthritis; Synovial Fluid; Transforming Growth Factor beta

Autophagy constitutes a defense mechanism to overcome aging and apoptosis in osteoarthritic cartilage. Several cytokines and transcription factors are linked to autophagy and play an important role in the degradative cascade in osteoarthritis (OA). Cell therapy such as platelet rich plasma (PRP) has recently emerged as a promising therapeutic tool for many diseases including OA. However, its mechanism of action on improving cartilage repair remains to be determined. The purpose of this study is to investigate the effect of PRP on osteoarthritic chondrocytes and to elucidate the mechanism by which PRP contributes to cartilage regeneration.. Osteoarthritic chondrocytes were co-cultured with an increasing concentration of PRP obtained from healthy donors. The effect of PRP on the proliferation of chondrocytes was performed using cell counting and WST8 proliferation assays. Autophagy, apoptosis and intracellular level of IL-4, IL-10, and IL-13 were determined using flow cytometry analyses. Autophagy markers BECLIN and LC3II were also determined using quantitative polymerase chain reaction (qPCR). qPCR and ELISA were used to measure the expression of ADAMDTS-5, MMP3, MMP13, TIMP-1-2-3, aggregan, Collagen type 2, TGF-β, Cox-2, Il-6, FOXO1, FOXO3, and HIF-1 in tissues and co-cultured media.. PRP increased significantly the proliferation of chondrocytes, decreased apoptosis and increased autophagy and its markers along with its regulators FOXO1, FOXO3 and HIF-1 in osteoarthritic chondrocytes. Furthermore, PRP caused a dose-dependent significant decrease in MMP3, MMP13, and ADAMTS-5, IL-6 and COX-2 while increasing TGF-β, aggregan, and collagen type 2, TIMPs and intracellular IL-4, IL-10, IL-13.. These results suggest that PRP could be a potential therapeutic tool for the treatment of OA.

Topics: Adult; Anti-Inflammatory Agents; Apoptosis; Autophagy; Blotting, Western; Cartilage, Articular; Case-Control Studies; Cell Proliferation; Cells, Cultured; Chondrocytes; Female; Humans; Interleukin-10; Interleukin-13; Interleukin-4; Male; Matrix Metalloproteinase 13; Matrix Metalloproteinase 3; Middle Aged; Osteoarthritis; Platelet-Rich Plasma; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta; Young Adult

Cross-talk between synovial fibroblasts (SF) and immune cells is suggested to play a crucial role in inflammation and chronification of rheumatoid arthritis (RA). The contribution of B cells in this process is poorly defined.. Here, primary B cells from healthy donors were polyclonally activated and cocultured with SF of non-synovitic origin from patients with osteoarthritis.. In B-SF cocultures the concentrations of interleukin 6 (IL-6) and IL-8 increased manifold compared with single cultures even under physical separation and remained stable for several days after B-cell removal. Intracellular staining confirmed SF as key producers of IL-6 and IL-8, and B cells as main producers of tumour necrosis factor alpha (TNFα) and IL-1ß. Blocking experiments with a combination of anti-TNFα-antibodies and rIL-1RA significantly reduced SF cytokine production by up to 90%, suggesting that B-cell-derived TNFα and IL-1ß were crucial mediators of SF activation. Interestingly, B-cell cytokine production, CD25 expression and proliferation decreased in cocultures by at least 50%, demonstrating a negative regulatory loop towards the activated B cells. Inhibition of activin receptor-like kinase 5, a crucial component of the tumour growth factor ß (TGFß) signalling pathway, partly restored B-cell proliferation, suggesting a contribution of SF-derived TGFß in B-cell suppression. Besides cytokines, B-cell-activated SF also upregulated secretion of matrix metalloproteases such as MMP-3, thereby acquiring potential tissue destructive properties. This was confirmed by their invasion into human cartilage in the severe combined immunodeficiency mouse fibroblast invasion model in vivo.. Interaction with activated B cells leads to conversion of non-arthritic SF into SF with a proinflammatory and aggressive RA-like phenotype, thereby suggesting a new, so far unrecognised role for B cells in RA pathogenesis.

Topics: Animals; Arthritis, Rheumatoid; B-Lymphocytes; Cartilage, Articular; Coculture Techniques; Cytokines; Fibroblasts; Heterografts; Humans; Immune Tolerance; Inflammation Mediators; Interleukin-1beta; Lymphocyte Activation; Matrix Metalloproteinases; Mice, SCID; Osteoarthritis; Signal Transduction; Synovial Fluid; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Examine whether osteoarthritis (OA) progression can be delayed by halofuginone in anterior cruciate ligament transection (ACLT) rodent models.. 3-month-old male C57BL/6J (wild type; WT) mice and Lewis rats were randomised to sham-operated, ACLT-operated, treated with vehicle, or ACLT-operated, treated with halofuginone. Articular cartilage degeneration was graded using the Osteoarthritis Research Society International (OARSI)-modified Mankin criteria. Immunostaining, flow cytometry, RT-PCR and western blot analyses were conducted to detect relative protein and RNA expression. Bone micro CT (μCT) and CT-based microangiography were quantitated to detect alterations of microarchitecture and vasculature in tibial subchondral bone.. Halofuginone attenuated articular cartilage degeneration and subchondral bone deterioration, resulting in substantially lower OARSI scores. Specifically, we found that proteoglycan loss and calcification of articular cartilage were significantly decreased in halofuginone-treated ACLT rodents compared with vehicle-treated ACLT controls. Halofuginone reduced collagen X (Col X), matrix metalloproteinase-13 and A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS 5) and increased lubricin, collagen II and aggrecan. In parallel, halofuginone-attenuated uncoupled subchondral bone remodelling as defined by reduced subchondral bone tissue volume, lower trabecular pattern factor (Tb.pf) and increased thickness of subchondral bone plate compared with vehicle-treated ACLT controls. We found that halofuginone exerted protective effects in part by suppressing Th17-induced osteoclastic bone resorption, inhibiting Smad2/3-dependent TGF-β signalling to restore coupled bone remodelling and attenuating excessive angiogenesis in subchondral bone.. Halofuginone attenuates OA progression by inhibition of subchondral bone TGF-β activity and aberrant angiogenesis as a potential preventive therapy for OA.

Topics: Animals; Anterior Cruciate Ligament; Bone and Bones; Bone Remodeling; Bone Resorption; Cartilage, Articular; Disease Models, Animal; Disease Progression; Male; Mice; Mice, Inbred C57BL; Osteoarthritis; Osteoclasts; Piperidines; Quinazolinones; Random Allocation; Rats; Rats, Inbred Lew; Transforming Growth Factor beta

Transforming growth factor-β (TGF-β) has been demonstrated as a potential therapeutic target in osteoarthritis. However, beneficial effects of TGF-β supplement and inhibition have both been reported, suggesting characterization of the spatiotemporal distribution of TGF-β during the whole time course of osteoarthritis is important. To investigate the activity of TGF-β in osteoarthritis progression, we collected knee joints from Dunkin-Hartley (DH) guinea pigs at 3, 6, 9, and 12-month old (n = 8), which develop spontaneous osteoarthritis in a manner extraordinarily similar to humans. Via histology and micro-computed tomography (CT) analysis, we found that the joints exhibited gradual cartilage degeneration, subchondral plate sclerosis, and elevated bone remodeling during aging. The degenerating cartilage showed a progressive switch of the expression of phosphorylated Smad2/3 to Smad1/5/8, suggesting dual roles of TGF-β/Smad signaling during chondrocyte terminal differentiation in osteoarthritis progression. In subchondral bone, we found that the locations and age-related changes of osterix(+) osteoprogenitors were in parallel with active TGF-β, which implied the excessive osteogenesis may link to the activity of TGF-β. Our study, therefore, suggests an association of cartilage degeneration and excessive bone remodeling with altered TGF-β signaling in osteoarthritis progression of DH guinea pigs. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:763-770, 2016.

Topics: Aging; Animals; Bone and Bones; Bone Marrow; Bone Remodeling; Cartilage, Articular; Female; Guinea Pigs; Male; Osteoarthritis; Smad Proteins; Transforming Growth Factor beta

Osteoarthritis (OA) is the most prevalent and debilitating joint disease, and there are currently no effective disease-modifying treatments available. Multiple risk factors for OA, such as aging, result in progressive damage and loss of articular cartilage. Autonomous circadian clocks have been identified in mouse cartilage, and environmental disruption of circadian rhythms in mice predisposes animals to OA-like damage. However, the contribution of the cartilage clock mechanisms to the maintenance of tissue homeostasis is still unclear. Here, we have shown that expression of the core clock transcription factor BMAL1 is disrupted in human OA cartilage and in aged mouse cartilage. Furthermore, targeted Bmal1 ablation in mouse chondrocytes abolished their circadian rhythm and caused progressive degeneration of articular cartilage. We determined that BMAL1 directs the circadian expression of many genes implicated in cartilage homeostasis, including those involved in catabolic, anabolic, and apoptotic pathways. Loss of BMAL1 reduced the levels of phosphorylated SMAD2/3 (p-SMAD2/3) and NFATC2 and decreased expression of the major matrix-related genes Sox9, Acan, and Col2a1, but increased p-SMAD1/5 levels. Together, these results define a regulatory mechanism that links chondrocyte BMAL1 to the maintenance and repair of cartilage and suggest that circadian rhythm disruption is a risk factor for joint diseases such as OA.

Topics: Animals; ARNTL Transcription Factors; Cartilage, Articular; Chondrocytes; Circadian Rhythm; Homeostasis; Humans; Male; Mice; Mice, Knockout; NFATC Transcription Factors; Osteoarthritis; Sequence Analysis, RNA; Transforming Growth Factor beta

Dickkopf-3 (Dkk3) is a non-canonical member of the Dkk family of Wnt antagonists and its upregulation has been reported in microarray analysis of cartilage from mouse models of osteoarthritis (OA). In this study we assessed Dkk3 expression in human OA cartilage to ascertain its potential role in chondrocyte signaling and cartilage maintenance.. Dkk3 expression was analysed in human adult OA cartilage and synovial tissues and during chondrogenesis of ATDC5 and human mesenchymal stem cells. The role of Dkk3 in cartilage maintenance was analysed by incubation of bovine and human cartilage explants with interleukin-1β (IL1β) and oncostatin-M (OSM). Dkk3 gene expression was measured in cartilage following murine hip avulsion. Whether Dkk3 influenced Wnt, TGFβ and activin cell signaling was assessed in primary human chondrocytes and SW1353 chondrosarcoma cells using qRT-PCR and luminescence assays.. Increased gene and protein levels of Dkk3 were detected in human OA cartilage, synovial tissue and synovial fluid. DKK3 gene expression was decreased during chondrogenesis of both ATDC5 cells and humans MSCs. Dkk3 inhibited IL1β and OSM-mediated proteoglycan loss from human and bovine cartilage explants and collagen loss from bovine cartilage explants. Cartilage DKK3 expression was decreased following hip avulsion injury. TGFβ signaling was enhanced by Dkk3 whilst Wnt3a and activin signaling were inhibited.. We provide evidence that Dkk3 is upregulated in OA and may have a protective effect on cartilage integrity by preventing proteoglycan loss and helping to restore OA-relevant signaling pathway activity. Targeting Dkk3 may be a novel approach in the treatment of OA.

Topics: Adaptor Proteins, Signal Transducing; Adult; Cartilage, Articular; Cells, Cultured; Chemokines; Chondrogenesis; Dose-Response Relationship, Drug; Down-Regulation; Humans; Intercellular Signaling Peptides and Proteins; Osteoarthritis; RNA, Small Interfering; Signal Transduction; Tissue Culture Techniques; Transforming Growth Factor beta; Up-Regulation; Wnt Signaling Pathway

Osteoarthritis (OA) is a major source of pain and disability worldwide with no effective medical therapy due to poor understanding of its pathogenesis. Transforming growth factor β (TGF-β) has been reported to play a role in subchondral bone pathology and articular cartilage degeneration during the progression of OA. In this study, we demonstrated that systemic use of a TGF-β-neutralizing antibody (1D11) attenuates OA progression by targeting subchondral bone pathological features in rodent OA models. Systemic administration of 1D11 preserves the subchondral bone microarchitecture, preventing articular cartilage degeneration by inhibition of excessive TGF-β activity, in both subchondral bone and the circulation. Moreover, the aberrant increases in the numbers of blood vessels, nestin(+) mesenchymal stromal/stem cells, and osterix(+) osteoblast progenitors were normalized by 1D11 systemic injection. Thus, systemic neutralization of excessive TGF-β ligands effectively prevented OA progression in animal models, with promising clinical implications for OA treatment.

Topics: Animals; Anterior Cruciate Ligament Injuries; Antibodies, Neutralizing; Bone and Bones; Cartilage; Disease Progression; Injections; Mice, Inbred C57BL; Neutralization Tests; Osteoarthritis; Osteogenesis; Transforming Growth Factor beta

Direct administration of therapeutic candidate gene sequences using the safe and effective recombinant adeno-associated virus (rAAV) vectors is a promising strategy to stimulate the biologic activities of articular chondrocytes as an adapted tool to treat human osteoarthritic (OA) cartilage. In the present study, we developed a combined gene transfer approach based on the co-delivery of the pleiotropic transformation growth factor beta (TGF-β) with the specific transcription factor SOX9 via rAAV to human normal and OA chondrocytes in vitro and cartilage explants in situ in light of the mitogenic and pro-anabolic properties of these factors. Effective, durable co-overexpression of TGF-β and SOX9 significantly enhanced the levels of cell proliferation both in human normal and OA chondrocytes and cartilage explants over an extended period of time (21 days), while stimulating the biosynthesis of key matrix components (proteoglycans, type-II collagen) compared with control conditions (reporter lacZ gene transfer, absence of vector treatment). Of further note, expression of hypertrophic type-X collagen significantly decreased following co-treatment by the candidate vectors. The present findings show the value of combining the transfer and expression of potent candidate factors in human OA cartilage as a means to re-establish essential features of normal cartilage and counteract the pathological shift of homeostasis. These observations support the concept of developing dual therapeutic rAAV gene transfer strategies as future, adapted tools for the direct treatment of human OA. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:2181-2190, 2016.

Topics: Aged; Cartilage, Articular; Cell Proliferation; Dependovirus; Genetic Therapy; Humans; Osteoarthritis; SOX9 Transcription Factor; Transforming Growth Factor beta; Transgenes

Peroxisome proliferator-activated receptor is closely associated with the pathogenesis of osteoarthritis. The level of exogenous advanced glycation end-products (AGEs) in articular cartilage is highly associated with the severity of osteoarthritic lesions. However, their interactions and role in promoting osteoarthritisprogression remain unclear. Here, we investigated the effect of AGEs on transforming growth factor (TGF)-β and matrix metalloproteinase (MMP)-9 expression, and discussed the correlation between AGEs and osteoarthritis, possible signaling pathways and mechanism in rabbit chondrocytes. TGF-β and MMP-9 mRNA and protein expression, catalase (CAT) and superoxide dismutase (SOD) activity, and malondialdehyde (MDA) and reactive oxygen species (ROS) levels were analyzed in chondrocytes treated with different concentrations of AGEs using RT-PCR and/or western blot; we detected NF-κB nuclear translocation by immunofluorescence. AGE treatment significantly increased TGF-β and MMP-9 mRNA and protein expression compared to controls (P < 0.01) in a dose-dependent manner (highest at 100 μg/mL). AGE-induced TGF-β and MMP-9 expressions in chondrocytes were significantly inhibited by anti-RAGE and PDTC (0.1 mM) treatment (P < 0.01). Furthermore, AGE-treatment significantly decreased CAT and SOD activity and increased MDA levels in a concentration-dependent manner compared to controls (P < 0.05), significantly promoting NF-κB nuclear translocation. AGE significantly inhibited the increased expression of TGF-β and MMP- 9, and induced chondrocyte damage. Its mechanism is associated with RAGE activation, increased ROS expression, and activation of the NF- κB signaling pathways.

Topics: Animals; Cartilage, Articular; Cells, Cultured; Chondrocytes; Down-Regulation; Glycation End Products, Advanced; Male; Matrix Metalloproteinase 13; Matrix Metalloproteinase 9; NF-kappa B; Osteoarthritis; PPAR alpha; Rabbits; Reactive Oxygen Species; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta

Joint trauma, which is frequently related with mechanical overloading of articular cartilage, is a well-established risk for osteoarthritis (OA) development. Additionally, reports show that trauma leads to synovial joint inflammation. In consequence, after joint trauma, cartilage is influenced by deleterious excessive loading combined with the catabolic activity of proinflammatory mediators. Since the activation of TGF-β signaling by loading is considered to be a key regulatory pathway for maintaining cartilage homeostasis, we tested the effect of proinflammatory conditions on mechanically mediated activation of TGF-β/Smad2/3P signaling in cartilage.. Cartilage explants were subjected to dynamic mechanical compression in the presence of interleukin-1 beta (IL-1β) or osteoarthritic synovium-conditioned medium (OAS-CM). Subsequently, the activation of the Smad2/3P pathway was monitored with QPCR analysis of reporter genes and additionally the expression of receptors activating the Smad2/3P pathway was analyzed. Finally, the ability for mechanically mediated activation of Smad2/3P was tested in human OA cartilage.. IL-1β presence during compression did not impair the upregulation of Smad2/3P reporter genes, however the results were affected by IL-1β-mediated upregulations in unloaded controls. OAS-CM significantly impaired the compression-mediated upregulation of bSmad7 and Tgbfb1. IL-1β suppressed the compression-mediated bAlk5 upregulation where 12 MPa compression applied in the presence of OAS-CM downregulated the bTgfbr2. Mechanically driven upregulation of Smad2/3P reporter genes was present in OA cartilage.. Proinflammatory conditions partly impair the mechanically mediated activation of the protective TGF-β/Smad2/3P pathway. Additionally, the excessive mechanical compression, applied in the presence of proinflammatory conditions diminishes the expression of the type II TGF-β receptor, a receptor critical for maintenance of articular cartilage.

Topics: Animals; Cartilage, Articular; Cattle; Chondrocytes; Gene Expression Profiling; Humans; Inflammation; Organ Culture Techniques; Osteoarthritis; Polymerase Chain Reaction; Signal Transduction; Smad Proteins; Stress, Mechanical; Transcriptome; Transforming Growth Factor beta

Epigenetic changes (i.e., chromatin modifications) occur during chondrogenesis and in osteoarthritis (OA). We investigated the effect of H3K27me3 demethylase inhibition on chondrogenesis and assessed its utility in cartilage tissue engineering and in understanding cartilage destruction in OA.. We used a high-content screen to assess the effect of epigenetic modifying compounds on collagen output during chondrogenesis of monolayer human mesenchymal stem cells (MSCs). The impact of GSK-J4 on gene expression, glycosaminoglycan output and collagen formation during differentiation of MSCs into cartilage discs was investigated. Expression of lysine (K)-specific demethylase 6A (UTX) and Jumonji domain-containing 3 (JMJD3), the HEK27Me3 demethylases targeted by GSK-J4, was measured in damaged and undamaged cartilage from patients with OA. The impact of GSK-J4 on ex vivo cartilage destruction and expression of OA-related genes in human articular chondrocytes (HACs) was assessed. H3K27Me3 demethylase regulation of transforming growth factor (TGF)-β-induced gene expression was measured in MSCs and HACs.. Treatment of chondrogenic MSCs with the H3K27me3 demethylase inhibitor GSK-J4, which targets JMJD3 and UTX, inhibited collagen output; expression of chondrogenic genes, including SOX9 and COL2A1; and disrupted glycosaminoglycan and collagen synthesis. JMJD3 but not UTX expression was increased during chondrogenesis and in damaged OA cartilage, suggesting a predominant role of JMJD3 in chondrogenesis and OA. GSK-J4 prevented ex vivo cartilage destruction and expression of the OA-related genes MMP13 and PTGS2. TGF-β is a key regulator of chondrogenesis and articular cartilage homeostasis, and TGF-β-induced gene expression was inhibited by GSK-J4 treatment of both chondrogenic MSCs and HACs.. Overall, we show that H3K27me3 demethylases modulate chondrogenesis and that enhancing this activity may improve production of tissue-engineered cartilage. In contrast, targeted inhibition of H3K27me3 demethylases could provide a novel approach in OA therapeutics.

Topics: Benzazepines; Cartilage, Articular; Cell Differentiation; Chondrocytes; Chondrogenesis; Enzyme Inhibitors; Gene Knockdown Techniques; Humans; Immunohistochemistry; In Vitro Techniques; Jumonji Domain-Containing Histone Demethylases; Mesenchymal Stem Cells; Microscopy, Electron, Scanning; Osteoarthritis; Pyrimidines; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta

Topics: Animals; Cohort Studies; Disease Models, Animal; Disease Progression; Gene Expression Regulation; Humans; Mice; Mice, Knockout; Osteoarthritis; Sensitivity and Specificity; Signal Transduction; Transforming Growth Factor beta

ADAMTS-4, also called aggrecanase 1, is considered to play a key role in aggrecan degradation in human osteoarthritic (OA) cartilage, but information about regulators of ADAMTS-4 aggrecanase activity remains limited. We undertook this study to search for molecules that modulate ADAMTS-4 activity.. Molecules copurified with ADAMTS-4 from ADAMTS-4-transfected chondrocytic cells were sequenced by nanoscale liquid chromatography tandem mass spectrometry. Binding activity was determined by immunoprecipitation and solid-phase binding assay. Effects on ADAMTS-4 activity were examined by aggrecan digestion assay. Expression of the binding molecule in OA cartilage and chondrocytes was examined by immunohistochemistry and reverse transcription-polymerase chain reaction.. We identified CCN1 (Cyr61) as an ADAMTS-4-binding protein and showed specific binding to the ADAMTS-4 cysteine-rich domain. Aggrecanase activity of ADAMTS-4 was inhibited by interaction with CCN1. Expression of messenger RNA for CCN1 was significantly higher in human OA cartilage than in normal cartilage. CCN1 was immunolocalized to chondrocytes in OA cartilage, showing direct correlations of immunoreactivity with the Mankin score of cartilage lesions and chondrocyte cloning. CCN1 and ADAMTS-4 were commonly coexpressed in clustered chondrocytes. CCN1 expression in OA chondrocytes was down-regulated by interleukin-1α (IL-1α) and up-regulated by transforming growth factor β (TGFβ). ADAMTS-4 expression was induced by treatment with IL-1α or TGFβ, but aggrecanase activity was detected only under stimulation with IL-1α. TGFβ-treated chondrocytes exhibited aggrecanase activity when CCN1 expression was knocked down.. Our findings provide the first evidence that CCN1 suppresses ADAMTS-4 activity and that CCN1 overexpression is directly correlated with chondrocyte cloning in OA cartilage. Our results suggest that the TGFβ/CCN1 axis plays a role in chondrocyte cluster formation through inhibition of ADAMTS-4.

Topics: ADAM Proteins; ADAMTS4 Protein; Aged; Aged, 80 and over; Cartilage, Articular; Case-Control Studies; Chondrocytes; Cysteine-Rich Protein 61; Gene Expression Profiling; Humans; Immunoprecipitation; Middle Aged; Osteoarthritis; Procollagen N-Endopeptidase; Protein Binding; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta

Ift88 is an intraflagella transport protein, critical for the cilium, and has been shown to be required for the maintenance of chondrocytes and cartilage. However, how Ift88 is controlled by cytokines that play a role in osteoarthritis is not well understood. Therefore, we examined the effects of TGF-β on the expression of Ift88. We used ATDC5 cells as chondrocytes and analyzed the effects of TGF-β on gene expression. TGF-β treatment suppresses the levels of Ift88 mRNA in a dose-dependent manner starting from as low as 0.5 ng/mL and reaching the nadir at around 2 ng/mL. TGF-β treatment also suppresses the protein levels of Ift88. TGF-β suppression of Ift88 is still observed when the cells are cultured in the presence of a transcriptional inhibitor while the TGF-β suppression is weakened in the presence of a protein synthesis inhibitor, cycloheximide. TGF-β treatment suppresses the levels of Ift88 mRNA stability suggesting the presence of posttranscriptional regulation. TGF-β treatment reduces the number of cilia positive cells and suppresses average length of cilia. Knockdown of Ift88 by siRNA enhances TGF-β-induced increase in type II collagen mRNA expression in ATDC5 cells revealing the suppressive role of Ift88 on TGF-β-induced regulation of extracellular matrix protein expression. TGF-β also suppresses Ift88 mRNA expression in primary culture of rib chondrocytes. These data indicate that TGF-β regulates Ift88 gene expression at least in part via posttrascriptional manner.

Topics: Animals; Cartilage, Articular; Chondrocytes; Cilia; Gene Expression Regulation; Mice; Osteoarthritis; Phosphorylation; Signal Transduction; Transforming Growth Factor beta; Tumor Suppressor Proteins

Human mesenchymal stromal cells (hMSC) differentiating toward the chondrogenic lineage recapitulate successive phases of embryonic chondrocyte maturation developing from progenitor cells to hypertrophic chondrocytes. Osteoarthritic cartilage is characterized by an alteration in chondrocyte metabolism and upregulation of hypertrophic differentiation markers. A number of studies point toward a functional role for microRNAs (miRs) in controlling chondrocyte differentiation and development of osteoarthritis (OA). However, information on miRs that may regulate a specific phase of chondrocyte maturation, especially hypertrophy, is lacking. We here aimed to unravel miR profiles modulated during chondrogenesis of hMSC to obtain new differentiation markers and potential new targets relevant for differentiation outcome and OA development. hMSC were subjected to transforming growth factor-β (TGF-β)-driven chondrogenesis and miR profiles were determined by microarray analysis at distinct developmental time points. Expression of selected miRs was compared to cultures lacking chondrogenesis and to redifferentiated nonhypertrophic articular chondrocytes. Among 1349 probed miRs, 553 were expressed and 169 (31%) were significantly regulated during chondrogenesis. Hierarchical clustering identified specific miR expression patterns representative for MSC, prechondrocytes, chondroblasts, chondrocytes, and hypertrophic chondrocytes, respectively. Regulation of miR-181 family members allowed discrimination of successive differentiation stages. Levels of several miRs, including miR-23b, miR-140, miR-181, and miR-210 positively correlated with successful chondrocyte formation. Hypertrophic MSC-derived chondrocytes and nonhypertrophic articular chondrocytes showed differential expression of miR-181a, miR-210, and miR-31, but not miR-148a implicated in COL10A1-regulation. We conclude that the here identified stage-dependent miR clusters may have imperative functions during chondrocyte differentiation providing novel diagnostic tools and targets of potential relevance for OA development.

Topics: Adult; Aged; Aged, 80 and over; Cell Differentiation; Chondrocytes; Collagen Type X; Female; Gene Expression Regulation; Humans; Hypertrophy; Male; MicroRNAs; Middle Aged; Osteoarthritis; Transforming Growth Factor beta

Lysyl hydroxylase 2b (LH2b) is known to increase pyridinoline cross-links, making collagen less susceptible to enzymatic degradation. Previously, we observed a relationship between LH2b and osteoarthritis-related fibrosis in murine knee joint. For this study, we investigate if transforming growth factor-beta (TGF-ß) and connective tissue growth factor (CTGF) regulate procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) (gene encoding LH2b) and LH2b expression differently in osteoarthritic human synovial fibroblasts (hSF). Furthermore, we investigate via which TGF-ß route (Smad2/3P or Smad1/5/8P) LH2b is regulated, to explore options to inhibit LH2b during fibrosis. To answer these questions, fibroblasts were isolated from knee joints of osteoarthritis patients. The hSF were stimulated with TGF-ß with or without a kinase inhibitor of ALK4/5/7 (SB-505124) or ALK1/2/3/6 (dorsomorphin). TGF-ß, CTGF, constitutively active (ca)ALK1 and caALK5 were adenovirally overexpressed in hSF. The gene expression levels of PLOD1/2/3, CTGF and COL1A1 were analyzed with Q-PCR. LH2 protein levels were determined with western blot. As expected, TGF-ß induced PLOD2/LH2 expression in hSF, whereas CTGF did not. PLOD1 and PLOD3 were not affected by either TGF-ß or CTGF. SB-505124 prevented the induction of TGF-ß-induced PLOD2, CTGF and COL1A1. Surprisingly, dorsomorphin completely blocked the induction of CTGF and COL1A1, whereas TGF-ß-induced PLOD2 was only slightly reduced. Overexpression of caALK5 in osteoarthritic hSF significantly induced PLOD2/LH2 expression, whereas caALK1 had no effect. We showed, in osteoarthritic hSF, that TGF-ß induced PLOD2/LH2 via ALK5 Smad2/3P. This elevation of LH2b in osteoarthritic hSF makes LH2b an interesting target to interfere with osteoarthritis-related persistent fibrosis.

Topics: Cells, Cultured; Connective Tissue Growth Factor; Fibroblasts; Fibrosis; Gene Expression Regulation; Humans; Osteoarthritis; Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad1 Protein; Smad2 Protein; Transforming Growth Factor beta

Emerging evidence has implied that subchondral bone plays an important role during osteoarthritis (OA) pathology. This study was undertaken to investigate whether abnormalities of the condylar subchondral bone lead to temporomandibular joint (TMJ) OA. We used an osteoblast-specific mutant TGF-β1 transgenic mouse, the CED mouse, in which high levels of active TGF-β1 occur in bone marrow, leading to abnormal bone remodeling. Subchondral bone changes in the mandibular condyles were investigated by micro-CT, and alterations in TMJ condyles were confirmed by histopathological and immunohistochemical analysis. Abnormalities in the condylar subchondral bone, characterized as fluctuant bone mineral density and microstructure and increased but uncoupled activity of osteoclasts and osteoblasts, were apparent in the 1- and 4-month CED mouse groups, while obvious cartilage degradation, in the form of cell-free regions and proteoglycan loss, was observed in the 4-month CED group. In addition, increased numbers of apoptotic chondrocytes and MMP9- and VEGF-positive chondrocytes were observed in the condylar cartilage in the 4-month CED group, but not in the 1-month CED group, compared with their respective age-matched controls. This study demonstrated that progressive degradation of mandibular condylar cartilage could be induced by the abnormal remodeling of the underlying subchondral bone during TMJOA progression.

Topics: Animals; Apoptosis; Bone Density; Bone Marrow; Bone Remodeling; Cartilage; Case-Control Studies; Caspase 3; Chondrocytes; Collagen Type I; Disease Models, Animal; Gene Expression Regulation; Mandibular Condyle; Matrix Metalloproteinase 13; Matrix Metalloproteinase 9; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Mice, Transgenic; Osteoarthritis; Osteoblasts; Osteoclasts; Proteoglycans; Temporomandibular Joint Disorders; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; X-Ray Microtomography

Synovial fibrosis is a major contributor to joint stiffness in osteoarthritis (OA). Transforming growth factor β (TGFβ), which is elevated in OA, plays a key role in the onset and persistence of synovial fibrosis. However, blocking of TGFβ in OA as a therapeutic intervention for fibrosis is not an option since TGFβ is crucial for cartilage maintenance and repair. Therefore, we undertook the present study to seek targets downstream of TGFβ for preventing OA-related fibrosis without interfering with joint homeostasis.. Experiments were performed to determine whether genes involved in extracellular matrix turnover were responsive to TGFβ and were elevated in OA-related fibrosis. We analyzed gene expression in TGFβ-stimulated human OA synovial fibroblasts and in the synovium of mice with TGFβ-induced fibrosis, mice with experimental OA, and humans with end-stage OA. Gene expression was determined by microarray, low-density array, or quantitative polymerase chain reaction analysis.. We observed an increase in expression of procollagen genes and genes encoding collagen crosslinking enzymes under all of the OA-related fibrotic conditions investigated. Comparison of gene expression in TGFβ-stimulated human OA synovial fibroblasts, synovium from mice with experimental OA, and synovium from humans with end-stage OA revealed that the genes PLOD2, LOX, COL1A1, COL5A1, and TIMP1 were up-regulated in all of these conditions. Additionally, we confirmed that these genes were up-regulated by TGFβ in vivo in mice with TGFβ-induced synovial fibrosis.. Most of the up-regulated genes identified in this study would be poor targets for therapy development, due to their crucial functions in the joint. However, the highly up-regulated gene PLOD2, responsible for the formation of collagen crosslinks that make collagen less susceptible to enzymatic degradation, is an attractive and promising target for interference in OA-related synovial fibrosis.

Topics: Animals; Arthritis, Experimental; Cartilage; Collagen; Fibrosis; Gene Expression; Humans; Mice; Osteoarthritis; Synovial Membrane; Transforming Growth Factor beta; Up-Regulation

Osteoarthritis (OA) is characterized by remodeling and degradation of joint tissues. Microarray studies have led to a better understanding of the molecular changes that occur in tissues affected by conditions such as OA; however, such analyses are limited to the identification of a list of genes with altered transcript expression, usually at a single time point during disease progression. While these lists have identified many novel genes that are altered during the disease process, they are unable to identify perturbed relationships between genes and gene products. In this work, we have integrated a time course gene expression dataset with network analysis to gain a better systems level understanding of the early events that occur during the development of OA in a mouse model. The subnetworks that were enriched at one or more of the time points examined (2, 4, 8, and 16 weeks after induction of OA) contained genes from several pathways proposed to be important to the OA process, including the extracellular matrix-receptor interaction and the focal adhesion pathways and the Wnt, Hedgehog and TGF-β signaling pathways. The genes within the subnetworks were most active at the 2 and 4 week time points and included genes not previously studied in the OA process. A unique pathway, riboflavin metabolism, was active at the 4 week time point. These results suggest that the incorporation of network-type analyses along with time series microarray data will lead to advancements in our understanding of complex diseases such as OA at a systems level, and may provide novel insights into the pathways and processes involved in disease pathogenesis.

Topics: Animals; Arthritis, Experimental; Disease Models, Animal; Disease Progression; Focal Adhesions; Gene Expression; Gene Expression Profiling; Hedgehog Proteins; Joints; Male; Metabolic Networks and Pathways; Mice; Mice, Inbred C57BL; Oligonucleotide Array Sequence Analysis; Osteoarthritis; Receptors, Cytoadhesin; Riboflavin; Transforming Growth Factor beta; Wnt Proteins; Wnt Signaling Pathway

Age is the most important risk factor for primary osteoarthritis (OA). Members of the TGF-β superfamily play a crucial role in chondrocyte differentiation and maintenance of healthy articular cartilage.. We have investigated whether age-related changes in TGF-β superfamily signaling components play a role in the relationship between OA-related cartilage degradation and aging.. The relationship between age, OA and TGF-β superfamily signaling was studied using murine experimental OA models, aging mice, bovine articular cartilage and human OA cartilage. The effects of TGF-β on cartilage homeostasis was studied with immunohistochemistry, Q-RT-PCR and signaling pathway analysis with Western blotting and the application of specific TGF-β inhibitors.. We have found that TGF-β loses its protective effects in old cartilage. Moreover, we found that on chondrocytes, TGF-β not only signals via the canonical type I receptor ALK5 (TGFBR1) but also via the ALK1 (ACVRL1) receptor. Remarkably, signaling via ALK5 (Smad2/3 route) results in protective while ALK1 signaling (Smad1/5/8 route) results in deleterious responses in articular chondrocytes. In cartilage of aging mice it was detected that the ALK1/ALK5 ratio is significantly increased, favoring TGF-β signaling via the Smad1/5/8 route, inducing changes in chondrocyte differentiation and matrix metalloproteinase-13 (MMP-13) expression. Moreover, human OA cartilage showed a significant correlation between ALK1 and MMP-13 expression. Since in mice aging and OA in often goes hand in hand, we also analyzed age-related expression of TGF-β superfamily related signaling molecules in healthy bovine cartilage in an age range from 6 months to 14 years. In this cohort of aging cartilage, we found that mainly signaling receptors determining the Smad2/3 pathway were decreased with age while Smad1/5/8-related signaling molecules did not alter, confirming our findings in aging mice.. Old cartilage appears to be less protected by TGF-β and shows significant alterations in TGF-β signaling pathways. Loss of the protective Smad2/3 pathway during aging can provide an explanation for the relationship between OA and aging.

Topics: Activin Receptors, Type II; Aging; Animals; Cartilage Diseases; Cartilage, Articular; Cattle; Chondrocytes; Humans; Interleukin-1; Matrix Metalloproteinase 13; Mice; Osteoarthritis; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta

The development of osteoarthritis (OA) depends on genetic and environmental factors, which influence the biology of the chondrocyte via epigenetic regulation. Changes within the epigenome might lead the way to discovery of new pathogenetic pathways. We performed a genome-wide methylation screening to identify potential differences between paired mild and severe osteoarthritic human cartilage. Sixteen female patients suffering from OA underwent total knee joint replacement. Cartilage specimens collected from corresponding macroscopically undamaged and from damaged areas were processed for DNA extraction and histology to evaluate the histological grading of the disease. Paired specimens were analysed for the methylation status of the whole genome using human promoter microarrays (Agilent, Santa Clara, CA). Selected target genes were then validated via methylation-specific qPCR. One thousand two hundred and fourteen genetic targets were identified differentially methylated between mild and severe OA. One thousand and seventy of these targets were found hypermethylated and 144 hypomethylated. The descriptive analysis of these genes by Gene Ontology (GO), KEGG pathway and protein domain analyses points to pathways of development and differentiation. We identified a list of genes which are differently methylated in mild and severe OA cartilage. Within the pathways of growth and development new therapeutic targets might arise by improving our understanding of pathogenetic mechanisms in OA.

Topics: Aged; Bone Morphogenetic Protein 7; Cartilage; DNA Methylation; Epigenesis, Genetic; Female; Humans; Middle Aged; Osteoarthritis; Polymerase Chain Reaction; Promoter Regions, Genetic; Transforming Growth Factor beta; Wnt Signaling Pathway

Osteoarthritis is a highly prevalent and debilitating joint disorder. There is no effective medical therapy for the condition because of limited understanding of its pathogenesis. We show that transforming growth factor β1 (TGF-β1) is activated in subchondral bone in response to altered mechanical loading in an anterior cruciate ligament transection (ACLT) mouse model of osteoarthritis. TGF-β1 concentrations are also high in subchondral bone from humans with osteoarthritis. High concentrations of TGF-β1 induced formation of nestin-positive mesenchymal stem cell (MSC) clusters, leading to formation of marrow osteoid islets accompanied by high levels of angiogenesis. We found that transgenic expression of active TGF-β1 in osteoblastic cells induced osteoarthritis, whereas inhibition of TGF-β activity in subchondral bone attenuated the degeneration of articular cartilage. In particular, knockout of the TGF-β type II receptor (TβRII) in nestin-positive MSCs led to less development of osteoarthritis relative to wild-type mice after ACLT. Thus, high concentrations of active TGF-β1 in subchondral bone seem to initiate the pathological changes of osteoarthritis, and inhibition of this process could be a potential therapeutic approach to treating this disease.

Topics: Animals; Bone and Bones; Cartilage; Cells, Cultured; Male; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; Osteoarthritis; Protein Serine-Threonine Kinases; Rats; Rats, Inbred Lew; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

Topics: Animals; Bone and Bones; Male; Mesenchymal Stem Cells; Osteoarthritis; Signal Transduction; Transforming Growth Factor beta

Topics: Animals; Bone and Bones; Male; Mesenchymal Stem Cells; Osteoarthritis; Signal Transduction; Transforming Growth Factor beta

MicroRNAs (miRNAs) have been shown to be important regulators in the pathogenesis of osteoarthritis (OA). The objective of this study was to determine the expression levels of miR-483, miR-483*, their host gene (Igf2) and other cytokines in a murine model of OA. The expression of miR-483 was significantly up-regulated in old mouse and in all of the operation groups, particularly the group assessed 1 week after surgery. The expression of miR-483* was significantly increased in the old mouse group and the group assessed 5 weeks after surgery. The expression of miR-483 was negatively correlated with the expression of (mRNA) Bmp7 and TgfB and positively correlated with Mmp13 by Pearson correlation analysis, while miR-483* was positively correlated with Il1B. Surprisingly, there was no correlation between the expression of either miR-483 or miR-483* and Igf2. This study shows that the expression of miR-483 and miR-483* is up-regulated in murine OA. These data suggest that miR-483 and miR-483* may play critical roles in early and later pathogenesis of OA, respectively, without the involvement of their host gene Igf2.

Topics: Animals; Bone Morphogenetic Protein 7; Cartilage; Cytokines; Disease Models, Animal; Immunohistochemistry; Insulin-Like Growth Factor II; Interleukin-1beta; Male; Matrix Metalloproteinase 13; Mice; MicroRNAs; Osteoarthritis; Transforming Growth Factor beta

Non-coding RNAs have been less studied in cartilage development and destruction regulated by sophisticated molecular events despite their considerable theranostic potential. In this study, we identified significant down-regulation of mR-101 and up-regulation of lncRNA, HOTTIP in the processes of endochondral ossification and osteoarthritic progression. In wing mesenchymal cells, up-expression of miR-101 by TGF-β3 treatment is targeting DNMT-3B and thereby altered the methylation of integrin-α1 addressed as a positive regulator of endochondral ossification in this study. In like manner, down-regulation of miR-101 also coordinately up-regulated DNMT-3B, down-regulated integrin-α1, and resulted in cartilage destruction. In an OA animal model, introduction of lentiviruses that encoded miR-101 or integrin-α1 successfully reduced cartilage destruction. In like manner, long non-coding RNA (lncRNA), HOTTIP, a known regulator for HoxA genes, was highly up-regulated and concurrent down-regulation of HoxA13 displayed the suppression of integrin-α1 in OA chondrocytes. In conclusion, two non-coding RNAs, miR-101 and HOTTIP regulate cartilage development and destruction by modulating integrin-α1 either epigenetically by DNMT-3B or transcriptionally by HoxA13 and data further suggest that these non-coding RNAs could be a potent predictive biomarker for OA as well as a therapeutic target for preventing cartilage-related diseases.

Topics: Animals; Cartilage; Cell Differentiation; Cells, Cultured; Chickens; Chondrocytes; DNA (Cytosine-5-)-Methyltransferases; DNA Methyltransferase 3B; Epigenesis, Genetic; Gene Expression Regulation; Homeodomain Proteins; Humans; Integrin alpha1; Male; Mesenchymal Stem Cells; Mice; MicroRNAs; Osteoarthritis; RNA, Antisense; RNA, Long Noncoding; Transforming Growth Factor beta

Therapeutic gene transfer is of significant value to elaborate efficient, durable treatments against human osteoarthritis (OA), a slow, progressive, and irreversible disorder for which there is no cure to date.. Here, we directly applied a recombinant adeno-associated virus (rAAV) vector carrying a human transforming growth factor beta (TGF-β) gene sequence to primary human normal and OA chondrocytes in vitro and cartilage explants in situ to monitor the stability of transgene expression and the effects of the candidate pleiotropic factor upon the regenerative cellular activities over time.. Efficient, prolonged expression of TGF-β achieved via rAAV gene transfer enhanced both the proliferative, survival, and anabolic activities of cells over extended periods of time in all the systems evaluated (at least for 21 days in vitro and for up to 90 days in situ) compared with control (reporter) vector delivery, especially in situ where rAAV-hTGF-β allowed for a durable remodeling of OA cartilage. Notably, sustained rAAV production of TGF-β in OA cartilage advantageously reduced the expression of key OA-associated markers of chondrocyte hypertrophic and terminal differentiation (type-X collagen, MMP-13, PTHrP, β-catenin) while increasing that of protective TIMPs and of the TGF-β receptor I in a manner that restored a favorable ALK1/ALK5 balance. Of note, the levels of activities in TGF-β-treated OA cartilage were higher than those of normal cartilage, suggesting that further optimization of the candidate treatment (dose, duration, localization, presence of modulating co-factors) will most likely be necessary to reproduce an original cartilage surface in relevant models of experimental OA in vivo without triggering potentially adverse effects.. The present findings show the ability of rAAV-mediated TGF-β gene transfer to directly remodel human OA cartilage by activating the biological, reparative activities and by regulating hypertrophy and terminal differentiation in damaged chondrocytes as a potential treatment for OA or for other disorders of the cartilage that may require transplantation of engineered cells.

Topics: Aged; Apoptosis; Cartilage, Articular; Cell Proliferation; Chondrocytes; Dependovirus; Humans; Osteoarthritis; Protective Agents; Recombination, Genetic; Signal Transduction; Transduction, Genetic; Transforming Growth Factor beta

MicroRNAs (miRNAs) down-regulate their target genes. The intronic miR-140, present in the WW domain containing E3 ubiquitin protein ligase 2 (WWP2) gene, decreases the expression of genes that play detrimental roles in osteoarthritis (OA). As the expression level of miR-140 is significantly decreased in human OA chondrocytes, we investigated its regulation in those cells.. Gene expression in human chondrocytes was determined by quantitative polymerase chain reaction (qPCR) and gene silencing was done in OA chondrocytes by transient transfection with specific small interfering RNAs (siRNAs). Binding sites of the miR-140 regulatory sequence (rsmiR-140) were identified by mutagenesis and chromatin immunoprecipitation (ChIP) in OA chondrocytes. The effects of translocation on OA chondrocytes were determined by immunocytochemistry and qPCR.. In contrast to miR-140, the expression of WWP2 was similar in both normal and OA cells, suggesting that miR-140 has an additional level of regulation. rsmiR-140 showed activity and predicted binding sites for nuclear matrix transcription factor 4 (NMP4), myc-associated zinc (MAZ), nuclear factor of activated T-cells (NFAT), and mothers against decapentaplegic homolog 3 (SMAD3). Silencing NFAT3 (P ≤0.01) and SMAD3 (P ≤0.05) differentially regulated miR-140 independently of WWP2. Silencing NFAT5 decreased both miR-140 and WWP2 (P ≤0.003 and P ≤0.05, respectively). NFAT3 activation increased and transforming growth factor-β (TGF-β) decreased rsmiR-140 activity. Mutagenesis of rsmiR-140 and ChIP assays identified binding sites at which NFAT3 (activator) and SMAD3 (repressor) directly regulated miR-140. TGF-β interfered with NFAT3 translocation, and subsequently with miR-140 expression.. This is the first study to provide evidence of a regulatory mechanism of miR-140 independent of WWP2, and new and differential roles for NFAT3 and SMAD3 in the OA process in the regulation of miR-140 transcription. Such knowledge could advance therapeutic strategies targeting OA.

Topics: Blotting, Western; Cells, Cultured; Chromatin Immunoprecipitation; Gene Expression Regulation; Gene Knockdown Techniques; Humans; Immunohistochemistry; MicroRNAs; NFATC Transcription Factors; Osteoarthritis; Real-Time Polymerase Chain Reaction; RNA, Small Interfering; Smad3 Protein; Transfection; Transforming Growth Factor beta; Ubiquitin-Protein Ligases

Articular cartilage has a very limited intrinsic repair capacity leading to progressive joint damage. Therapies involving tissue engineering depend on chondrogenic differentiation of progenitor cells. This chondrogenic differentiation will have to survive in a diseased joint. We postulate that catabolic factors in this environment inhibit chondrogenesis of progenitor cells. We investigated the effect of a catabolic environment on chondrogenesis in pellet cultures of human mesenchymal stem cells (hMSCs). We exposed chondrogenically differentiated hMSC pellets, to interleukin (IL)-1α, tumor necrosis factor (TNF)-α or conditioned medium derived from osteoarthritic synovium (CM-OAS). IL-1α and TNF-α in CM-OAS were blocked with IL-1Ra or Enbrel, respectively. Chondrogenesis was determined by chondrogenic markers collagen type II, aggrecan, and the hypertrophy marker collagen type X on mRNA. Proteoglycan deposition was analyzed by safranin o staining on histology. IL-1α and TNF-α dose-dependently inhibited chondrogenesis when added at onset or during progression of differentiation, IL-1α being more potent than TNF-α. CM-OAS inhibited chondrogenesis on mRNA and protein level but varied in extent between patients. Inhibition of IL-1α partially overcame the inhibitory effect of the CM-OAS on chondrogenesis whereas the TNF-α contribution was negligible. We show that hMSC chondrogenesis is blocked by either IL-1α or TNF-α alone, but that there are additional factors present in CM-OAS that contribute to inhibition of chondrogenesis, demonstrating that catabolic factors present in OA joints inhibit chondrogenesis, thereby impairing successful tissue engineering.

Topics: Aged; Bone Morphogenetic Protein 2; Cell Differentiation; Chondrogenesis; Culture Media, Conditioned; Female; Humans; Interleukin-1alpha; Male; Mesenchymal Stem Cells; Middle Aged; Osteoarthritis; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Class I phosphoinositide 3 kinase (PI3K) δ is a promising therapeutic target for rheumatoid arthritis (RA) because of its contribution to leukocyte biology. However, its contribution in fibroblasts has not been studied as a mechanism that contributes to efficacy. We investigated the expression and function of PI3Kδ in synovium and cultured fibroblast-like synoviocytes (FLS). Immunohistochemistry demonstrated that PI3Kδ is highly expressed in RA synovium, especially in the synovial lining. Using quantitative PCR and Western blot analysis, we found that PI3Kδ mRNA and protein expression is higher in RA than in osteoarthritis (OA) synovium. PI3Kδ was also expressed in cultured FLS, along with PI3Kα and PI3Kβ, whereas PI3Kγ was not detectable. PI3Kδ mRNA expression was selectively induced by inflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) but not by growth factors platelet-derived growth factor (PDGF) and transforming growth factor β (TGFβ). The use of inhibitors that block individual PI3K isoforms, including the novel selective PI3Kδ inhibitor INK007, showed that PI3Kδ is required for PDGF- and TNF-induced Akt activation. PI3Kδ inhibition also diminished PDGF-mediated synoviocyte growth and sensitized cells to H(2)O(2)-induced apoptosis. These data are the first documentation of increased PI3Kδ expression in both RA synovium and cultured synoviocytes. Furthermore, these are the first data demonstrating that PI3Kδ is a major regulator of PDGF-mediated fibroblast growth and survival via Akt. Thus, targeting PI3Kδ in RA could modulate synoviocyte function via anti-inflammatory and disease-altering mechanisms.

Topics: Apoptosis; Arthritis, Rheumatoid; Cell Division; Cell Survival; Cells, Cultured; Class I Phosphatidylinositol 3-Kinases; Cytokines; Enzyme Inhibitors; Fibroblasts; Gene Expression Regulation, Enzymologic; Humans; Inflammation Mediators; Osteoarthritis; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-akt; RNA, Messenger; Synovial Membrane; Transforming Growth Factor beta

Lead remains a significant environmental toxin, and we believe we may have identified a novel target of lead toxicity in articular chondrocytes. These cells are responsible for the maintenance of joint matrix, and do so under the regulation of TGF-β signaling. As lead is concentrated in articular cartilage, we hypothesize that it can disrupt normal chondrocyte phenotype through suppression of TGF-β signaling. These experiments examine the effects of lead exposure in vivo and in vitro at biologically relevant levels, from 1 nM to 10 µM on viability, collagen levels, matrix degrading enzyme activity, TGF-β signaling, and articular surface morphology. Our results indicate that viability was unchanged at levels ≤100 µM Pb, but low and high level lead in vivo exposure resulted in fibrillation and degeneration of the articular surface. Lead treatment also decreased levels of type II collagen and increased type X collagen, in vivo and in vitro. Additionally, MMP13 activity increased in a dose-dependent manner. Active caspase 3 and 8 were dose-dependently elevated, and treatment with 10 µM Pb resulted in increases of 30% and 500%, respectively. Increasing lead treatment resulted in a corresponding reduction in TGF-β reporter activity, with a 95% reduction at 10µM. Levels of phosphoSmad2 and 3 were suppressed in vitro and in vivo and lead dose-dependently increased Smurf2. These changes closely parallel those seen in osteoarthritis. Over time this phenotypic shift could compromise maintenance of the joint matrix.

Topics: Animals; Cartilage, Articular; Cell Line; Chickens; Chondrocytes; Lead; Osteoarthritis; Phenotype; Rats; Signal Transduction; Toxicity Tests, Acute; Transforming Growth Factor beta

Recent studies have implied that osteoarthritis (OA) is a metabolic disease linked to deregulation of genes involved in lipid metabolism and cholesterol efflux. Sterol Regulatory Element Binding Proteins (SREBPs) are transcription factors regulating lipid metabolism with so far no association with OA. Our aim was to test the hypothesis that SREBP-2, a gene that plays a key role in cholesterol homeostasis, is crucially involved in OA pathogenesis and to identify possible mechanisms of action.. We performed a genetic association analysis using a cohort of 1,410 Greek OA patients and healthy controls and found significant association between single nucleotide polymorphism (SNP) 1784G>C in SREBP-2 gene and OA development. Moreover, the above SNP was functionally active, as normal chondrocytes' transfection with SREBP-2-G/C plasmid resulted in interleukin-1β and metalloproteinase-13 (MMP-13) upregulation. We also evaluated SREBP-2, its target gene 3-hydroxy-3-methylglutaryl-coenzymeA reductase (HMGCR), phospho-phosphoinositide3-kinase (PI3K), phospho-Akt, integrin-alphaV (ITGAV) and transforming growth factor-β (TGF-β) mRNA and protein expression levels in osteoarthritic and normal chondrocytes and found that they were all significantly elevated in OA chondrocytes. To test whether TGF-β alone can induce SREBP-2, we treated normal chondrocytes with TGF-β and found significant upregulation of SREBP-2, HMGCR, phospho-PI3K and MMP-13. We also showed that TGF-β activated aggrecan (ACAN) in chondrocytes only through Smad3, which interacts with SREBP-2. Finally, we examined the effect of an integrin inhibitor, cyclo-RGDFV peptide, on osteoarthritic chondrocytes, and found that it resulted in significant upregulation of ACAN and downregulation of SREBP-2, HMGCR, phospho-PI3K and MMP-13 expression levels.. We demonstrated, for the first time, the association of SREBP-2 with OA pathogenesis and provided evidence on the molecular mechanism involved. We suggest that TGF-β induces SREBP-2 pathway activation through ITGAV and PI3K playing a key role in OA and that integrin blockage may be a potential molecular target for OA treatment.

Topics: Adult; Aged; Aged, 80 and over; Aggrecans; Alleles; Cells, Cultured; Chondrocytes; Collagen Type I; Collagen Type II; Female; Gene Expression Regulation; Genetic Predisposition to Disease; Genotype; Heterozygote; Humans; Hydroxycholesterols; Hydroxymethylglutaryl CoA Reductases; Integrin alphaV; Male; Middle Aged; Oligopeptides; Osteoarthritis; Peptides, Cyclic; Phenotype; Phosphatidylinositol 3-Kinases; Phosphorylation; Polymorphism, Single Nucleotide; Protein Binding; Proto-Oncogene Proteins c-akt; Receptors, Transforming Growth Factor beta; Smad3 Protein; Sterol Regulatory Element Binding Protein 2; Transforming Growth Factor beta

The mechanism by which intra-articular injection of hyaluronan (HA) ameliorates joint pathology is unknown. Animal studies have shown that HA can reduce synovial activation, periarticular fibrosis and cartilage erosion; however, its specific effects on the different cell types involved remain unclear. We have used the TTR (TGFbeta1 injection and Treadmill Running) model of murine osteoarthritis (OA), which exhibits many OA-like changes, including synovial activation, to examine in vivo tissue-specific effects of intra-articular HA.. The kinetics of clearance of fluorotagged HA from joints was examined with whole-body imaging. Naïve and treated knee joints were examined macroscopically for cartilage erosion, meniscal damage and fibrosis. Quantitative histopathology was done with Safranin O for cartilage and with Hematoxylin & Eosin for synovium. Gene expression in joint tissues for Acan, Col1a1, Col2a1, Col3a1, Col5a1, Col10a1, Adamts5 and Mmp13 was done by quantitative PCR. The abundance and distribution of aggrecan, collagen types I, II, III, V and X, ADAMTS5 and MMP13 were examined by immunohistochemistry.. Injected HA showed a half-life of less than 2 h in the murine knee joint. At the tissue level, HA protected against neovascularization and fibrosis of the meniscus/synovium and maintained articular cartilage integrity in wild-type but not in Cd44 knockout mice. HA injection enhanced the expression of chondrogenic genes and proteins and blocked that of fibrogenic/degradative genes and proteins in cartilage/subchondral bone, whereas it blocked activation of both groups in meniscus/synovium. In all locations it reduced the expression/protein for Mmp13 and blocked Adamts5 expression but not its protein abundance in the synovial lining.. The injection of HA, 24 h after TGFbeta1 injection, inhibited the cascade of OA-like joint changes seen after treadmill use in the TTR model of OA. In terms of mechanism, tissue protection by HA injection was abrogated by Cd44 ablation, suggesting that interaction of the injected HA with CD44 is central to its protective effects on joint tissue remodeling and degeneration in OA progression.

Topics: ADAM Proteins; ADAMTS5 Protein; Animals; Cartilage, Articular; Disease Models, Animal; Extracellular Matrix Proteins; Fibrosis; Hyaluronan Receptors; Hyaluronic Acid; Immunohistochemistry; Injections, Intra-Arterial; Male; Matrix Metalloproteinase 13; Mice; Mice, Inbred C57BL; Mice, Knockout; Neovascularization, Pathologic; Osteoarthritis; Polymerase Chain Reaction; Synovial Membrane; Transcriptome; Transforming Growth Factor beta; Viscosupplements

This study evaluates cytokines production in bone and bone marrow of patients with an osteoporotic fracture or with osteoarthritis by real time PCR, Western blot and immunohistochemistry. We demonstrate that the cytokine pattern is shifted towards osteoclast activation and osteoblast inhibition in patients with osteoporotic fractures.. Fragility fractures are the resultant of low bone mass and poor bone architecture typical of osteoporosis. Cytokines involved in the control of bone cell maturation and function are produced by both bone itself and bone marrow cells, but the roles of these two sources in its control and the amounts they produce are not clear. This study compares their production in patients with an osteoporotic fracture and those with osteoarthritis.. We evaluated 52 femoral heads from women subjected to hip-joint replacement surgery for femoral neck fractures due to low-energy trauma (37), or for osteoarthritis (15). Total RNA was extracted from both bone and bone marrow, and quantitative PCR was used to identify the receptor activator of nuclear factor kB Ligand (RANKL), osteoprotegerin (OPG), macrophage colony stimulating factor (M-CSF), transforming growth factor β (TGFβ), Dickoppf-1 (DKK-1) and sclerostin (SOST) expression. Immunohistochemistry and Western blot were performed in order to quantify and localize in bone and bone marrow the cytokines.. We found an increase of RANKL/OPG ratio, M-CSF, SOST and DKK-1 in fractured patients, whereas TGFβ was increased in osteoarthritic bone. Bone marrow produced greater amounts of RANKL, M-CSF and TGFβ compared to bone, whereas the production of DKK-1 and SOST was higher in bone.. We show that bone marrow cells produced the greater amount of pro-osteoclastogenic cytokines, whereas bone cells produced higher amount of osteoblast inhibitors in patients with fragility fracture, thus the cytokine pattern is shifted towards osteoclast activation and osteoblast inhibition in these patients.

Topics: Adaptor Proteins, Signal Transducing; Aged; Aged, 80 and over; Blotting, Western; Bone Marrow; Bone Morphogenetic Proteins; Cytokines; Female; Femur Head; Genetic Markers; Humans; Intercellular Signaling Peptides and Proteins; Macrophage Colony-Stimulating Factor; Middle Aged; Osteoarthritis; Osteoblasts; Osteoclasts; Osteoporotic Fractures; Osteoprotegerin; RANK Ligand; Real-Time Polymerase Chain Reaction; Transforming Growth Factor beta

We and others have previously shown that IL-17 is elevated in the synovial fluid of patients with reactive arthritis (ReA)/undifferentiated spondyloarthropathy (uSpA) having acute synovitis. Major source for IL-17 is Th17 cells, which differentiate from Th0 cells under the influence of TGF-β and IL-6, IL1-β and are maintained by IL-21 and 23. There is a paucity of data on these cytokines in ReA/uSpA. Thus, we measured the levels of Th-17 differentiating and maintaining cytokines in synovial fluid of patients with ReA and uSpA. Fifty patients with ReA/uSpA (ReA 24, uSpA 26), 19 patients with rheumatoid arthritis (RA) and 11 patients with osteoarthritis (OA) were included in the study. Synovial fluid (SF) were collected from knee joint and stored at -80°C until analysis. Cytokines were assayed using ELISA in SF specimens. The median IL-17A levels were significantly elevated in ReA (48.3 pg/ml) and uSpA (32.5 pg/ml) as compared to non-inflammatory OA controls (<7.8 pg/ml; p < 0.0001), while comparable to RA (57.9 pg/ml). Further, IL-6 median values were higher in ReA (25.2 ng/ml) and uSpA (13.6 ng/ml) as compared to OA (0.76 ng/ml; p < 0.0001), and comparable to RA (15.8 ng/ml). The median levels of IL-1β, IL-21 levels were elevated in ReA, uSpA and RA as compared to OA but were not statistically significant. TGF-β levels in ReA and uSpA were similar to OA but lower than in RA (4340 pg/ml; p < 0.05). IL-23 was not detectable in any synovial fluid sample. However, levels of these cytokines did not correlate with disease activity parameters. Significant positive correlation was observed between IL-17 and IL-1β (r = 0.38, p < 0.005), IL-17 and IL-6 (r = 0.659, p < 0.0001), and IL-1β and IL-6 (r = 0.391, p < 0.0001) in ReA and uSpA group. Inflammatory synovitis in ReA/uSpA is mediated by pro-inflammatory cytokines like IL-17, IL-6, IL-1β, and IL-21. However, IL-23 was not detectable in SF. Good correlation between IL-17, IL-6, and IL 1β suggest that either they are co-regulated or they regulate each other.

Topics: Adult; Arthritis, Reactive; Arthritis, Rheumatoid; Case-Control Studies; Female; Humans; Interleukin-17; Interleukin-1beta; Interleukin-23; Interleukin-6; Interleukins; Male; Osteoarthritis; Prohibitins; Retrospective Studies; Spondylarthropathies; Synovial Fluid; Synovitis; Th17 Cells; Transforming Growth Factor beta

Thoracic aortic aneurysms and dissections are a main feature of connective tissue disorders, such as Marfan syndrome and Loeys-Dietz syndrome. We delineated a new syndrome presenting with aneurysms, dissections and tortuosity throughout the arterial tree in association with mild craniofacial features and skeletal and cutaneous anomalies. In contrast with other aneurysm syndromes, most of these affected individuals presented with early-onset osteoarthritis. We mapped the genetic locus to chromosome 15q22.2-24.2 and show that the disease is caused by mutations in SMAD3. This gene encodes a member of the TGF-β pathway that is essential for TGF-β signal transmission. SMAD3 mutations lead to increased aortic expression of several key players in the TGF-β pathway, including SMAD3. Molecular diagnosis will allow early and reliable identification of cases and relatives at risk for major cardiovascular complications. Our findings endorse the TGF-β pathway as the primary pharmacological target for the development of new treatments for aortic aneurysms and osteoarthritis.

Topics: Age of Onset; Aorta, Thoracic; Aortic Aneurysm; Chromosomes, Human, Pair 15; Family Health; Female; Humans; Immunohistochemistry; Male; Mutation; Osteoarthritis; Radiography; Signal Transduction; Smad3 Protein; Syndrome; Transforming Growth Factor beta

A large number of experiments have been performed to identify genetic loci that influence osteoarthritis (OA) susceptibility, with a particular focus on the primary form of the disease. Unfortunately, the currently reported candidate-gene, genome-wide linkage scans and genome-wide association scans have tended to highlight the heterogeneous nature of OA rather than generate statistically compelling genome signals. Nevertheless, some breakthroughs have been made. For example, genetic susceptibility within genes coding for components of the transforming growth factor β pathway has emerged as a particularly interesting find, while completely novel loci are also being uncovered, such as the signal to a cluster of genes on chromosome 7q22. It also appears that quantitative effects on gene expression, rather than qualitative effects on protein function, are particularly important, and that we need to consider the effects of genetic susceptibility in joint formation as much as we do in joint maintenance. Nevertheless, we are still only at the beginning of our search, and much more sophisticated clinical and laboratory approaches will need to be applied before we get a clear understanding of the genetic indicators that influence OA susceptibility.

Topics: Extracellular Matrix Proteins; Genetic Predisposition to Disease; Genome-Wide Association Study; Growth Differentiation Factor 5; Humans; Osteoarthritis; Smad3 Protein; Thyroid Hormones; Transforming Growth Factor beta

Cartilage matrix homeostasis involves a dynamic balance between numerous signals that modulate chondrocyte functions. This study aimed at elucidating the role of the extracellular glucose concentration in modulating anabolic and catabolic gene expression in normal and osteoarthritic (OA) human chondrocytes and its ability to modify the gene expression responses induced by pro-anabolic stimuli, namely Transforming Growth Factor-β (TGF). For this, we analyzed by real time RT-PCR the expression of articular cartilage matrix-specific and non-specific genes, namely collagen types II and I, respectively. The expression of the matrix metalloproteinases (MMPs)-1 and -13, which plays a major role in cartilage degradation in arthritic conditions, and of their tissue inhibitors (TIMP) was also measured. The results showed that exposure to high glucose (30 mM) increased the mRNA levels of both MMPs in OA chondrocytes, whereas in normal ones only MMP-1 increased. Collagen II mRNA was similarly increased in normal and OA chondrocytes, but the increase lasted longer in the later. Exposure to high glucose for 24 h prevented TGF-induced downregulation of MMP-13 gene expression in normal and OA chondrocytes, while the inhibitory effect of TGF on MMP-1 expression was only partially reduced. Other responses were not significantly modified. In conclusion, exposure of human chondrocytes to high glucose, as occurs in vivo in diabetes mellitus patients and in vitro for the production of engineered cartilage, favors the chondrocyte catabolic program. This may promote articular cartilage degradation, facilitating OA development and/or progression, as well as compromise the quality and consequent in vivo efficacy of tissue engineered cartilage.

Topics: Chondrocytes; Collagen Type I; Collagen Type II; Gene Expression; Glucose; Humans; Matrix Metalloproteinase 1; Matrix Metalloproteinase 13; Osteoarthritis; Transforming Growth Factor beta

A number of recent papers on the WWP2 E3 ubiquitin ligase and two novel WWP2 isoforms have revealed important biological insight and disease-specific functions, and also impacted on our understanding of ubiquitin ligases in cell cycle regulation, apoptosis and differentiation. Gene knockout studies suggest a developmental role for WWP2 in chondrogenesis via mechanisms involving cartilage-specific transcription factors. Furthermore, WWP2 isoforms have been shown to selectively target oncogenic signaling pathways linked to both the pTEN tumour suppressor and the TGFβ/Smad signaling pathway. Here, it is suggested that WWP2 isoforms have now emerged as central physiological regulators as well as promising new disease targets, and that the challenge ahead is to now develop highly selective WWP2 inhibitors with utility in cartilage disease such as osteoarthritis and as new anticancer strategies.

Topics: Cartilage Diseases; Chondrocytes; Chondrogenesis; Humans; Osteoarthritis; Protein Isoforms; PTEN Phosphohydrolase; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Ubiquitin-Protein Ligases

To investigate the relationship between inflammatory responses of the temporomandibular joint (TMJ) and the metacarpophalangeal (MCP) joint in clinically normal horses.. 7 mature horses.. In each horse, 1 TMJ and 1 MCP joint were injected with lipopolysaccharide (LPS; 0.0025 μg). The contralateral TMJ and MCP joint were injected with saline (0.9% NaCl) solution. Synovial fluid samples were collected from all 4 joints over 24 hours after injection. Concentrations of interleukin-6, tumor necrosis factor-α, transforming growth factor-β, and total protein were measured via immunoassay. Horses were assessed for clinical signs of joint inflammation at each time point.. Concentrations of interleukin-6 were not significantly different between LPS-injected MCP joints and TMJs at any time point. Transforming growth factor-β concentrations were significantly increased in MCP joints, compared with concentrations in TMJs, at 12 and 24 hours after injection. Tumor necrosis factor-α concentrations were significantly higher in LPS-injected TMJs than in LPS-injected MCP joints at 1 and 6 hours after injection. Total protein concentration did not differ significantly between LPS-injected MCP joints and TMJs. Injection of LPS induced clinical inflammation at all time points; additionally, 2 MCP joints (but no TMJs) had an inflammatory response to injection of saline solution.. The inflammatory response to LPS appeared to be attenuated more quickly in TMJs than in MCP joints of horses. The difference in response suggested that a lack of clinical osteoarthritis in the TMJ of horses could be attributable to a difference in cytokine response.

Topics: Animals; Cytokines; Female; Horse Diseases; Horses; Injections, Intra-Articular; Interleukin-6; Lipopolysaccharides; Male; Metacarpophalangeal Joint; Osteoarthritis; Proteins; Random Allocation; Synovial Fluid; Temporomandibular Joint; Time Factors; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

A series of new oxicam derivatives bearing a quaternary ammonium (QA) moiety was synthesized and evaluated in vitro for antiosteoarthritis properties. Propyltrimethyl ammonium 3 and propyldiethylmethyl ammonium 11 stimulated aggrecan expression and mitigated the inhibitory action of IL-1. QA derivative 3 also increased TGF-beta2 and type II receptor expression. These results suggest that such derivatives may not only inhibit the osteoarthritis degradation process but also stimulate its regeneration. QA derivatives 3 and 11 offer potential for developing new therapeutic approaches to osteoarthritis treatment.

Topics: Aggrecans; Animals; Cartilage, Articular; Cattle; Cells, Cultured; Chondrocytes; Gene Expression Regulation; Osteoarthritis; Quaternary Ammonium Compounds; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

Several recent in vitro investigations and experimental studies performed in animal models of osteoarthritis (OA) sustained the previously held view that interleukin (IL)-1 or tumour necrosis factor-alpha (TNFalpha) disrupt the metabolism of synovial joint tissues. The evidence to date indicates that, in addition to IL-1 and TNFalpha, other pro-inflammatory cytokines, including IL-6, members of the IL-6 protein superfamily, IL-7, IL-17 and IL-18, can also promote articular cartilage extracellular matrix protein degradation or synergize with other cytokines to amplify and accelerate cartilage destruction. Most importantly, many of these cytokines have been implicated in causing synovial tissue activation and damage to subchondral bone as well as altering cartilage homeostasis in spontaneously occurring or surgically induced animal models of OA and in transgenic mice genetically primed to develop OA. In this regard, these pro-inflammatory cytokines may also play a significant role in the pathogenesis of human OA. However, attempts to modify the progression of human OA in well designed, controlled clinical trials with an IL-1 receptor antagonist protein (IRAP) have not been successful. Several anabolic cytokines (also termed growth factors), including transforming growth factor-beta (TGF-beta), insulin-like growth factor-1 (IGF-1), fibroblast growth factor-2 (FGF-2), platelet-derived growth factor (PDGF) and connective tissue growth factor (CTGF), have also been proposed as regulators of skeletal long bone growth and development as well as cartilage and bone homeostasis. TGF-beta, IGF-1 and FGF-2, in particular, have been characterized as potential chondroprotective agents. Thus, enzymatic disruption and removal of these growth factors from cartilage extracellular matrix proteins, as in the case of TGF-beta and FGF-2, or disruption of their function, as in the case of the enhanced binding of free IGF-1 with IGF binding proteins in OA joint synovial fluid, may compromise and ultimately be responsible for the inadequate repair of articular cartilage in OA. An improved understanding of the cellular and molecular mechanisms by which pro-inflammatory and/or anabolic cytokines alter both the structure and function of synovial joints may eventually result in the commercial development of disease-modifying OA drugs (DMOADs). Since the prevalence of OA is high in the elderly population, future development of DMOADs must also take into account potential differences in

Topics: Aged; Antirheumatic Agents; Arthritis, Experimental; Cartilage; Cartilage, Articular; Cytokines; Fibroblast Growth Factor 2; Humans; Insulin-Like Growth Factor I; Interleukin 1 Receptor Antagonist Protein; Interleukin-1; Interleukin-17; Interleukin-18; Interleukin-1beta; Interleukin-6; Interleukin-7; Joints; Osteoarthritis; Platelet-Derived Growth Factor; Signal Transduction; Synovial Fluid; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

In osteoarthritis, angiogenesis, which occurs in the osteochondral junction and synovium, may accelerate inflammation and contribute to the severity of the disease. We used anterior cruciate ligament-transection (ACLT) to investigate the therapeutic effect of an angiogenesis inhibitor, thrombospondin-1 (TSP-1), in a rat model of osteoarthritis. Osteoarthritis was induced in Wistar rats in the knee of one hind leg. After ACLT, AdTSP-1 (adenoviral vector encoding mouse TSP-1) was intraarticularly injected into the knee joints. Transgene expression, angiogenesis, and inflammatory responses in the knee joints were examined. They were also assessed morphologically, radiographically, and histologically for manifestations of disease. The levels of TSP-1 peaked on day 3 and were substantially maintained for at least 9 days after AdTSP-1 infection. Adenovirus-mediated gene expression was detected in the synovial membrane and chondrocytes. TSP-1 gene transfer induced transforming growth factor-β (TGF-β) production, but it reduced microvessel density, macrophage infiltration, and interleukin-1β (IL-1β) levels. Gross morphological and histopathological examinations revealed that rats treated with AdTSP-1 had less severe osteoarthritis than controls. In vivo adenovirus-mediated TSP-1 gene transfer significantly reduced microvessel density, inflammation, and suppressed the progression of osteoarthritis. This study provides potential applications of TSP-1 gene delivery for treating osteoarthritis.

Topics: Adenoviridae; Animals; Cartilage, Articular; Disease Models, Animal; Disease Progression; Gene Transfer Techniques; Inflammation; Injections, Intra-Articular; Interleukin-1beta; Joints; Male; Neovascularization, Pathologic; Osteoarthritis; Radiography; Rats; Rats, Wistar; Thrombospondin 1; Transforming Growth Factor beta; Treatment Outcome

Transforming growth factor-β (TGF-β) plays a critical role in cartilage homeostasis and deregulation of its signalling is implicated in osteoarthritis (OA). TGF-β isoforms signal through a pair of transmembrane serine/threonine kinases known as the type I and type II TGF-β receptors. Endoglin is a TGF-β co-receptor that binds TGF-β with high affinity in the presence of the type II TGF-β receptor. We have previously shown that endoglin is expressed in human chondrocytes and that it forms a complex with the TGF-β signalling receptors. However, the functional significance of endoglin expression in chondrocytes is unknown. Our objective was to determine whether endoglin regulates TGF-β/Smad signalling and extracellular matrix (ECM) production in human chondrocytes and whether its expression varies with chondrocyte differentiation state.. Endoglin function was determined by overexpression or antisense morpholino/siRNA knockdown of endoglin in human chondrocytes and measuring TGF-β-induced Smad phosphorylation, transcriptional activity and ECM production. Alterations in endoglin expression levels were determined during subculture-induced dedifferentiation of human chondrocytes and in normal vs OA cartilage samples.. Endoglin enhances TGF-β1-induced Smad1/5 phosphorylation and inhibits TGF-β1-induced Smad2 phosphorylation, Smad3-driven transcriptional activity and ECM production in human chondrocytes. In addition, the enhancing effect of endoglin siRNA knockdown on TGF-β1-induced Smad3-driven transcription is reversed by ALK1 overexpression. Furthermore, endoglin levels are increased in chondrocytes following subculture-induced dedifferentiation and in OA cartilage as compared to normal cartilage.. Together, our results suggest that endoglin regulates the balance between TGF-β/ALK1/Smad1/5 and ALK5/Smad2/3 signalling and ECM production in human chondrocytes and that endoglin may represent a marker for chondrocyte phenotype.

Topics: Antigens, CD; Blotting, Western; Cartilage, Articular; Cell Differentiation; Cells, Cultured; Chondrocytes; Endoglin; Extracellular Matrix; Gene Expression Regulation; Humans; Immunohistochemistry; Osteoarthritis; Phosphorylation; Receptors, Cell Surface; Smad Proteins, Receptor-Regulated; Transforming Growth Factor beta

In osteoarthritis (OA) articular chondrocytes undergo phenotypic changes culminating in the progressive loss of cartilage from the joint surface. The molecular mechanisms underlying these changes are poorly understood. Here we report enhanced (approximately 7-fold) expression of F-spondin, a neuronal extracellular matrix glycoprotein, in human OA cartilage (P<0.005). OA-specific up-regulation of F-spondin was also demonstrated in rat knee cartilage following surgical menisectomy. F-spondin treatment of OA cartilage explants caused a 2-fold increase in levels of the active form of TGF-beta1 (P<0.01) and a 10-fold induction of PGE2 (P<0.005) in culture supernatants. PGE2 induction was found to be dependent on TGF-beta and the thrombospondin domain of the F-spondin molecule. F-spondin addition to cartilage explant cultures also caused a 4-fold increase in collagen degradation (P<0.05) and a modest reduction in proteoglycan synthesis (approximately 20%; P<0.05), which were both TGF-beta and PGE2 dependent. F-spondin treatment also led to increased secretion and activation of MMP-13 (P<0.05). Together these studies identify F-spondin as a novel protein in OA cartilage, where it may act in situ at lesional areas to activate latent TGF-beta and induce cartilage degradation via pathways that involve production of PGE2.

Topics: Animals; Cartilage, Articular; Cells, Cultured; Chondrocytes; Extracellular Matrix Proteins; Humans; Osteoarthritis; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta; Up-Regulation

The regeneration of diseased hyaline cartilage continues to be a great challenge, mainly because degeneration--caused either by major injury or by age-related processes--can overextend the tissue's self-renewal capacity. We show that repair tissue from human articular cartilage during the late stages of osteoarthritis harbors a unique progenitor cell population, termed chondrogenic progenitor cells (CPCs). These exhibit stem cell characteristics such as clonogenicity, multipotency, and migratory activity. The isolated CPCs, which exhibit a high chondrogenic potential, were shown to populate diseased tissue ex vivo. Moreover, downregulation of the osteogenic transcription factor runx-2 enhanced the expression of the chondrogenic transcription factor sox-9. This, in turn, increased the matrix synthesis potential of the CPCs without altering their migratory capacity. Our results offer new insights into the biology of progenitor cells in the context of diseased cartilage tissue. Our work may be relevant in the development of novel therapeutics for the later stages of osteoarthritis.

Topics: Bone Morphogenetic Protein 6; Cartilage, Articular; Cell Differentiation; Cell Movement; Chondrocytes; Chondrogenesis; Collagen Type II; Core Binding Factor Alpha 1 Subunit; Gene Knockdown Techniques; Humans; Osteoarthritis; Osteoblasts; Osteogenesis; Regeneration; RNA, Small Interfering; SOX9 Transcription Factor; Stem Cells; Transforming Growth Factor beta

Bone tissue in osteoarthritis (OA) is composed of abundant undermineralized osteoid matrix. The aim of this study was to investigate the mechanisms responsible for this abnormal matrix, using in vitro OA subchondral osteoblasts.. Primary normal and OA osteoblasts were prepared from tibial plateaus. Phenotype was determined by alkaline phosphatase activity, and osteocalcin, osteopontin, prostaglandin E2 (PGE2), and transforming growth factor beta1 (TGFbeta1) were assessed by enzyme-linked immunosorbent assay. Expression of COL1A1 and COL1A2 was determined by real-time polymerase chain reaction. The production of type I collagen was determined by the release of its C-terminal propeptide and Western blot analysis. In vitro mineralization was evaluated by alizarin red staining. Inhibition of TGFbeta1 expression was performed using a small interfering RNA technique.. Mineralization of OA osteoblasts was reduced compared with mineralization of normal osteoblasts, even in the presence of bone morphogenetic protein 2 (BMP-2). Alkaline phosphatase and osteocalcin levels were elevated in OA osteoblasts compared with normal osteoblasts, whereas osteopontin levels were similar. The COL1A1-to-COL1A2 messenger RNA ratio was 3-fold higher in OA osteoblasts compared with normal osteoblasts, and the production of collagen by OA osteoblasts was increased. Because TGFbeta1 inhibits BMP-2-dependent mineralization, and because TGFbeta1 levels are approximately 4-fold higher in OA osteoblasts than in normal osteoblasts, inhibiting TGFbeta1 levels in OA osteoblasts corrected the abnormal COL1A1-to-COL1A2 ratio and increased alizarin red staining.. Elevated TGFbeta1 levels in OA osteoblasts are responsible, in part, for the abnormal ratio of COL1A1 to COL1A2 and for the abnormal production of mature type I collagen. This abnormal COL1A1-to-COL1A2 ratio generates a matrix that blunts mineralization in OA osteoblasts.

Topics: Aged; Alkaline Phosphatase; Anthraquinones; Bone Morphogenetic Protein 2; Calcification, Physiologic; Cells, Cultured; Collagen; Collagen Type I; Collagen Type I, alpha 1 Chain; Coloring Agents; Dinoprostone; Extracellular Matrix Proteins; Female; Humans; Male; Osteoarthritis; Osteoblasts; Osteocalcin; Osteopontin; RNA, Messenger; Transforming Growth Factor beta

Similarly to humans, healthy, wild-type mice develop osteoarthritis, including of the temporomandibular joint (TMJ), as a result of aging. Pro-inflammatory cytokines, such as IL-1beta, IL-6, and TNFalpha, are known to contribute to the development of osteoarthritis, whereas TGFbeta has been associated with articular regeneration. We hypothesized that a balance between IL-1beta and TGFbeta underlies the development of TMJ osteoarthritis, whereby IL-1beta signaling down-regulates TGFbeta expression as part of disease pathology. Our studies in wild-type mice, as well as the Col1-IL1beta(XAT) mouse model of osteoarthritis, demonstrated an inverse correlation between IL-1beta and TGFbeta expression in the TMJ. IL-1beta etiologically correlated with joint pathology, whereas TGFbeta expression associated with IL-1beta down-regulation and improvement of articular pathology. Better understanding of the underlying inflammatory processes during disease will potentially enable us to harness inflammation for orofacial tissue regeneration.

Topics: Animals; Down-Regulation; Female; Interleukin-1beta; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Mice, Transgenic; Osteoarthritis; Signal Transduction; Temporomandibular Joint; Temporomandibular Joint Disorders; Transforming Growth Factor beta

This study aimed to examine the effect of injection of transforming growth factor beta (TGF-beta) on articular cartilage in osteoarthritic temporomandibular joint (TMJ).. Disc perforation was performed bilaterally in 24 rabbits to induce osteoarthritis (OA). The right joint was injected with TGF-beta as experimental groups, and the left joint with physiologic saline as control. Animals were killed at different intervals. Histology and reverse transcriptase polymerase chain reaction was performed for comparison.. All joints showed OA-like changes, but the degree in the experimental was significantly less severe than in the control. At 12 weeks a significantly greater expression of aggrecan and collagen type II was found in the experimental compared with the control joints. However, no difference in either anabolic or catabolic genes was found between the two groups at 24 weeks.. Transforming growth factor beta may have a potential benefit in protecting articular cartilage during the development of TMJ OA.

Topics: Aggrecans; Animals; Arthritis, Experimental; Cartilage, Articular; Collagen Type II; Endopeptidases; Fibrocartilage; Glyceraldehyde-3-Phosphate Dehydrogenases; Interleukin-1; Mandibular Condyle; Osteoarthritis; Rabbits; Temporomandibular Joint; Temporomandibular Joint Disorders; Time Factors; Transforming Growth Factor beta

To investigate the mechanism how Transforming growth factor-beta(TGF-beta) represses Interleukin-1beta (IL-1beta)-induced Proteinase-Activated Receptor-2 (PAR-2) expression in human primary synovial cells (hPSCs). Human chondrocytes and hPSCs isolated from cartilages and synovium of Osteoarthritis (OA) patients were cultured with 10% fetal bovine serum media or serum free media before treatment with IL-1beta, TGF-beta1, or Connective tissue growth factor (CTGF). The expression of PAR-2 was detected using reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting. Collagen zymography was performed to assess the activity of Matrix metalloproteinases-13 (MMP-13). It was demonstrated that IL-1beta induces PAR-2 expression via p38 pathway in hPSCs. This induction can be repressed by TGF-beta and was observed to persist for at least 48 hrs, suggesting that TGF-beta inhibits PAR-2 expression through multiple pathways. First of all, TGF-beta was able to inhibit PAR-2 activity by inhibiting IL-1beta-induced p38 signal transduction and secondly the inhibition was also indirectly due to MMP-13 inactivation. Finally, TGF-beta was able to induce CTGF, and in turn CTGF represses PAR-2 expression by inhibiting IL-1beta-induced phospho-p38 level. TGF-beta could prevent OA from progression with the anabolic ability to induce CTGF production to maintain extracellular matrix (ECM) integrity and to down regulate PAR-2 expression, and the anti-catabolic ability to induce Tissue inhibitors of metalloproteinase-3 (TIMP-3) production to inhibit MMPs leading to avoid PAR-2 over-expression. Because IL-1beta-induced PAR-2 expressed in hPSCs might play a significantly important role in early phase of OA, PAR-2 repression by exogenous TGF-beta or other agents might be an ideal therapeutic target to prevent OA from progression.

Topics: Collagen; Connective Tissue Growth Factor; Culture Media, Serum-Free; Disease Progression; Extracellular Matrix; Gene Expression Profiling; Gene Expression Regulation, Enzymologic; Humans; Interleukin-1beta; Osteoarthritis; p38 Mitogen-Activated Protein Kinases; Receptor, PAR-2; Signal Transduction; Synovial Membrane; Transforming Growth Factor beta

To establish a cell culture system for noninvasive and real-time monitoring of chondrogenic differentiation in order to screen for chondrogenic factors.. The optimum reporter construct transfected into chondrogenic ATDC5 cells was selected by a luciferase reporter assay and fluorescence analysis during cultures with insulin. The established cell line was validated according to its fluorescence following stimulation with SOX proteins, bone morphogenetic protein 2 (BMP-2), or transforming growth factor beta (TGFbeta) and was compared with the level of messenger RNA for COL2A1 as well as with the degree of Alcian blue staining. Screening of chondrogenic factors was performed by expression cloning using a retroviral expression library prepared from human tracheal cartilage. The expression pattern of the identified molecule was examined by in situ hybridization and immunohistochemistry. Functional analysis was performed by transfection of the identified gene, the small interfering RNA, and the mutated gene.. We established an ATDC5 cell line with 4 repeats of a highly conserved enhancer ligated to a COL2A1 basal promoter and the DsRed2 reporter (ATDC5-C2ER). Fluorescence was induced under the stimulations with SOX proteins, BMP-2, or TGFbeta, showing good correspondence to the chondrogenic markers. Screening using the ATDC5-C2ER system identified several chondrogenic factors, including sorting nexin 19 (SNX19). SNX19 was expressed in the limb cartilage of mouse embryos and in the degraded cartilage of adult mouse knee joints during osteoarthritis progression. The gain-of-function and loss-of-function analyses revealed a potent chondrogenic activity of SNX19.. We established the ATDC5-C2ER system for efficient monitoring of chondrogenic differentiation by fluorescence analysis, and we identified a novel chondrogenic factor (SNX19) using this system. This system will be useful for elucidating the molecular network of chondrogenic differentiation.

Topics: Animals; Bone Morphogenetic Protein 2; Carrier Proteins; Cartilage, Articular; Cell Dedifferentiation; Cell Line; Chondrocytes; Chondrogenesis; Collagen Type II; Disease Models, Animal; Disease Progression; Humans; Mice; Mice, Inbred C57BL; Osteoarthritis; Sorting Nexins; SOX Transcription Factors; Stem Cells; Transforming Growth Factor beta; Vesicular Transport Proteins

Rheumatoid arthritis (RA) is a chronic inflammatory and destructive joint disease characterized by overexpression of pro-inflammatory/pro-destructive genes and other activating genes (for example, proto-oncogenes) in the synovial membrane (SM). The gene expression in disease is often characterized by significant inter-individual variances via specific synchronization/desynchronization of gene expression. To elucidate the contribution of the variance to the pathogenesis of disease, expression variances were tested in SM samples of RA patients, osteoarthritis (OA) patients, and normal controls (NCs).. Analysis of gene expression in RA, OA, and NC samples was carried out using Affymetrix U133A/B oligonucleotide arrays, and the results were validated by real-time reverse transcription-polymerase chain reaction. For the comparison between RA and NC, 568 genes with significantly different variances in the two groups (P

Topics: Adult; Aged; Apoptosis Regulatory Proteins; Arthritis, Rheumatoid; Case-Control Studies; Cell Survival; Female; Gene Expression Profiling; Genetic Variation; Humans; Inflammation; Male; Middle Aged; Neovascularization, Pathologic; Osteoarthritis; Receptors, Cytokine; RNA, Messenger; Signal Transduction; Synovial Membrane; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

To optimize sampling and to understand sources of variation in biomarkers for osteoarthritis (OA), we evaluated variation due to activity and food consumption.. Twenty participants, with radiographic knee OA, provided serial serum and urine samples at 4 time points: before arising in the morning; after 1 h of light activity; 1 h after eating breakfast; and in the evening. Five serum (s) and 2 urinary (u) analytes were measured: hyaluronan (sHA); cartilage oligomeric matrix protein (sCOMP); keratan sulfate (sKS-5D4); transforming growth factor beta (sTGF-ss1); and collagen II-related epitopes (sCPII, uCTXII, and uC2C). Activity was monitored by an accelerometer.. All serum biomarkers increased and one of the urinary biomarkers decreased after 1 h of non-exertional activity. Food consumption following activity was associated with a return of biomarker concentrations to baseline levels. Accelerometers proved to be a novel way to monitor protocol compliance and demonstrated a positive association between the mean level of activity and sCOMP concentration. Urinary CTXII varied the least but demonstrated both true circadian variation (peak in the morning and nadir in the evening) and the most robust correlation with radiographic knee OA.. We confirm activity related variation in these markers. These data suggested that biomarkers also varied due to upright posture, glomerular filtration rate stimulated by food intake, and circadian rhythm in the case of uCTXII.

Topics: Aged; Aged, 80 and over; Biomarkers; Cartilage Oligomeric Matrix Protein; Circadian Rhythm; Collagen Type II; Eating; Epitopes; Extracellular Matrix Proteins; Female; Glycoproteins; Humans; Hyaluronic Acid; Keratan Sulfate; Knee; Male; Matrilin Proteins; Middle Aged; Motor Activity; Osteoarthritis; Radiography; Transforming Growth Factor beta

The mechanisms of osteoclast maturation and the role of rheumatoid arthritis (RA) synovial fibroblasts in the control of osteoclastogenesis remain unclear. The purpose of this study was to determine the humoral factors that influence osteoclast differentiation resulting from mutual interactions between osteoclast progenitor cells and synovial fibroblasts.. The cloned mouse macrophage cell line RAW 264.7 or isolated human CD14+ monocytes were cocultured with RA or osteoarthritis (OA) synovial fibroblasts in the presence of RANKL. Osteoclasts were visualized by staining for tartrate-resistant acid phosphatase (TRAP), and their functions were evaluated by bone resorption assay. Transforming growth factor beta (TGFbeta) and osteoprotegerin (OPG) levels were measured by enzyme-linked immunosorbent assay. Expression of pSmad2 and Smad7 was analyzed by Western blotting.. RANKL-mediated osteoclast formation was observed in cocultures of RAW cells with RA synovial cells, but not with OA synovial cells. This formation was inhibited by TGFbeta receptor kinase inhibitor or neutralizing TGFbeta antibody. Human CD14+ monocytes showed the same results with RAW 264.7, and bone resorption activity was consistent with osteoclast formation. RA synovial fibroblasts produced TGFbeta in response to cell-cell contact with RAW cells in a RANKL-dependent manner. TGFbeta reduced OPG production by RA synovial fibroblasts, but dose-dependently increased OPG secretion in OA synovial fibroblasts. TGFbeta decreased the expression of pSmad2 and increased the expression of Smad7 in RA synovial fibroblasts, but not OA synovial fibroblasts.. Suppression of OPG production by down-regulation of TGFbeta/Smad2 signaling may contribute to RANKL-mediated osteoclastogenesis from RA synovial fibroblasts.

Topics: Animals; Arthritis, Rheumatoid; Bone Resorption; Cell Differentiation; Cell Line; Cloning, Organism; Down-Regulation; Fibroblasts; Humans; Mice; Monocytes; Osteoarthritis; Osteoclasts; Osteoprotegerin; RANK Ligand; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad7 Protein; Synovial Membrane; Transforming Growth Factor beta

We showed previously that the attachment of synovial fibroblasts to laminin (LM)-111 in the presence of transforming growth factor-beta induces significant expression of the matrix metalloproteinase (MMP)-3. Here we go on to investigate the regulation of additional MMPs and their specific tissue inhibitors of matrix proteases (TIMPs). Changes in steady-state mRNA levels encoding TIMPs and MMPs were investigated by quantitative reverse transcription-polymerase chain reaction. Production of MMPs was monitored by a multiplexed immunoarray. Signal transduction pathways were studied by immunoblotting. Attachment of synovial fibroblasts to LM-111 in the presence of transforming growth factor-beta induced significant increases in MMP-3 mRNA (12.35-fold, p < 0.001) and protein (mean 62 ng/ml, sixfold, p < 0.008) and in expression of MMP-10 mRNA (11.68-fold, p < 0.05) and protein (54 ng/ml, 20-fold, p > or = 0.02). All other TIMPs and MMPs investigated failed to show this LM-111-facilitated transforming growth factor-beta response. No phosphorylation of nuclear factor-kappaB was observed. We conclude that co-stimulation of synovial fibroblasts by LM-111 together with transforming growth factor-beta suffices to induce significant expression of MMP-3 and MMP-10 by synovial fibroblasts and that this induction is independent of nuclear factor-kappaB phosphorylation.

Topics: Arthritis, Rheumatoid; Cells, Cultured; Early Growth Response Protein 1; Electrophoresis, Polyacrylamide Gel; Fibroblasts; Gene Expression Regulation; Humans; Laminin; Matrix Metalloproteinase 10; Matrix Metalloproteinase 3; NF-kappa B; Osteoarthritis; Phosphorylation; Proto-Oncogene Proteins c-fos; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Synovial Membrane; Tissue Inhibitor of Metalloproteinases; Transforming Growth Factor beta

Bone cells produce many glycoproteins potentially involved in the maintenance of healthy bone tissues. Two cytokines produced in inflamed joints, tumor necrosis factor (TNF)alpha and transforming growth factor (TGF)beta, have previously been shown to alter cellular glycosylation which may potentially affect the expression and function of glycoproteins. In order to evaluate models to study the glycodynamics of bone cells, we examined primary human osteoblastic cells from osteoarthritis patients, and compared these to human osteosarcoma cells MG63 and SJSA-1. We showed here for the first time that all of the human osteoblastic cells actively synthesize complex N- and O-glycan chains of bone cell glycoproteins, with quantitative differences between cell types. TNFalpha-induced apoptosis or TGFbeta-induced cell differentiation and proliferation had significant effects on both cell surface carbohydrates and glycosyltransferase activities of osteoblasts and osteosarcoma cells. The results indicate that cultured human bone-derived osteoblastic cells are good models to examine the glycodynamics of osteoblasts under conditions of cell growth and cell death. The changes induced by cytokines can result in altered cell surface functions which may be of importance in osteoarthritis, osteoporosis and other bone diseases.

Topics: Apoptosis; Bone and Bones; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cells, Cultured; Glycosylation; Humans; Osteoarthritis; Osteoblasts; Osteosarcoma; Polysaccharides; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

The purpose of this study was to determine whether osteogenic protein 1 (OP-1) would protect articular cartilage from degeneration during the development of osteoarthritis (OA) in the rabbit anterior cruciate ligament transection (ACLT) model. Previous studies have shown that OP-1 is vital to cartilage matrix integrity and repair, stimulates synthesis of cartilage matrix components, proteoglycans, and collagen, and has a protective effect against catabolic mediators like matrix metalloproteinases and interleukin-1.. The rabbit ACLT model was used in which the anterior cruciate ligament was transected leading to OA. OP-1 was delivered to the joint surgically for approximately 6 weeks by implantation of an Alzet osmotic pump into the medial thigh with a catheter threaded from the pump into the knee joint. Forty rabbits (20 control and 20 experimental) had the ACLT surgery and implantation of the pump performed simultaneously. They were sacrificed after 9 weeks for analysis. The OA was graded using the Outerbridge classification with India Ink staining. Histological staining and histomorphometry with Hematoxylin & Eosin and Safranin O were performed to analyze OA progression and semi-quantitative polymerase chain reaction (PCR) was performed for anabolic and catabolic genes.. The experimental group had an average Outerbridge score of 1.8 vs 2.5 for the controls (P<0.05). Histomorphometry showed 10.9% surface deterioration or an average depression of 0.05mm vs 22.3% and 0.1mm for the controls (P<0.05). Semi-quantitative PCR showed a significantly greater expression of aggrecan and collagen type II in the OP-1 treated cartilage when compared to controls and less expression of aggrecanase, a catabolic mediator.. OP-1 may have a potential benefit in protecting articular cartilage during the development of OA.

Topics: Aggrecans; Animals; Arthritis, Experimental; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Cartilage, Articular; Collagen Type II; Gene Expression; Matrix Metalloproteinases; Osteoarthritis; Rabbits; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Severity of Illness Index; Transforming Growth Factor beta

To investigate the potential of transgene-activated periosteal cells for permanently resurfacing large partial-thickness cartilage defects.. In miniature pigs, autologous periosteal cells stimulated ex vivo by bone morphogenetic protein 2 gene transfer, using liposomes or a combination of adeno-associated virus (AAV) and adenovirus (Ad) vectors, were applied on a bioresorbable scaffold to chondral lesions comprising the entire medial half of the patella. The resulting repair tissue was assessed, 6 and 26 weeks after transplantation, by histochemical and immunohistochemical methods. The biomechanical properties of the repair tissue were characterized by nanoindentation measurements. Implants of unstimulated cells and untreated lesions served as controls.. All grafts showed satisfactory integration into the preexisting cartilage. Six weeks after transplantation, AAV/Ad-stimulated periosteal cells had adopted a chondrocyte-like phenotype in all layers; the newly formed matrix was rich in proteoglycans and type II collagen, and its contact stiffness was close to that of healthy hyaline cartilage. Unstimulated periosteal cells and cells activated by liposomal gene transfer formed only fibrocartilaginous repair tissue with minor contact stiffness. However, within 6 months following transplantation, the AAV/Ad-stimulated cells in the superficial zone tended to dedifferentiate, as indicated by a switch from type II to type I collagen synthesis and reduced contact stiffness. In deeper zones, these cells retained their chondrocytic phenotype, coinciding with positive staining for type II collagen in the matrix.. Large partial-thickness cartilage defects can be resurfaced efficiently with hyaline-like cartilage formed by transgene-activated periosteal cells. The long-term stability of the cartilage seems to depend on physicobiochemical factors that are active only in deeper zones of the cartilaginous tissue.

Topics: Adenoviridae; Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cartilage Diseases; Cell Transplantation; Disease Models, Animal; Female; Genetic Therapy; Hyaline Cartilage; Models, Biological; Osteoarthritis; Periosteum; Swine; Swine, Miniature; Transforming Growth Factor beta; Transgenes; Transplantation, Autologous; Wound Healing

Growth factor therapy may be useful for stimulation of cartilage matrix synthesis and repair. Thus, the purpose of our study was to further understand the effect of combined insulin-like growth factor-1 (IGF-1) and osteogenic protein-1 (OP-1) treatment on the matrix synthesized by human adult normal and osteoarthritic (OA) chondrocytes.. Chondrocytes were isolated post-mortem from articular cartilage from tali of normal human donors and femoral condyles of OA patients undergoing knee replacement surgery. Cells were cultured in alginate beads for 21 days in four experimental groups: (1) "mini-ITS" control; (2) 100 ng/ml IGF-1; (3) 100 ng/ml OP-1; (4) IGF-1+OP-1, each at 100 ng/ml. Beads were processed for histological (Safranin O and fast green), morphometrical and immunohistochemical (aggrecan, decorin, type I, II, VI, and X collagens, and fibronectin accumulation) analyses.. Histology showed that IGF-1 alone did not induce substantial matrix production. OP-1 alone caused a considerable matrix formation, but the highest matrix accumulation by normal and OA chondrocytes was found when OP-1 and IGF-1 were added together. Morphometrical analysis indicated larger matrices produced by OA chondrocytes than by normal cells under the combined treatment. All tested matrix proteins were more abundant in the combination group. Type X collagen was detected only under the combined OP-1 and IGF-1 treatment and was present at very low levels. Type I collagen was found only in OA chondrocytes.. The results obtained in the current study suggest that combined therapy with IGF-1 and OP-1 may have a greater potential in treating cartilage defects seen in OA than use of either growth factor alone.

Topics: Alginates; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Cartilage, Articular; Cells, Cultured; Chondrocytes; Drug Synergism; Extracellular Matrix; Humans; Insulin-Like Growth Factor Binding Proteins; Intercellular Signaling Peptides and Proteins; Osteoarthritis; Transforming Growth Factor beta

Regulatory T cells (Tregs) exert their anti-inflammatory activity predominantly by cell contact-dependent mechanisms. A study was undertaken to investigate the regulatory capacity of autologous peripheral blood Tregs in contact with synovial tissue cell cultures, and to evaluate their presence in peripheral blood, synovial tissue and synovial fluid of patients with rheumatoid arthritis (RA).. 44 patients with RA and 5 with osteoarthritis were included in the study. The frequency of interferon (IFN)gamma-secreting cells was quantified in synovial tissue cell cultures, CD3-depleted synovial tissue cell cultures, synovial tissue cultures co-cultured with autologous CD4+ and with CD4+CD25+ peripheral blood T cells by ELISPOT. Total CD3+, Th1 polarised and Tregs were quantified by real-time PCR for CD3epsilon, T-bet and FoxP3 mRNA, and by immunohistochemistry for FoxP3 protein.. RA synovial tissue cell cultures exhibited spontaneous expression of IFNgamma which was abrogated by depletion of CD3+ T cells and specifically reduced by co-culture with autologous peripheral blood Treg. The presence of Treg in RA synovitis was indicated by FoxP3 mRNA expression and confirmed by immunohistochemistry. The amount of FoxP3 transcripts, however, was lower in the synovial membrane than in peripheral blood or synovial fluid. The T-bet/FoxP3 ratio correlated with both a higher grade of synovial tissue lymphocyte infiltration and higher disease activity.. This study has shown, for the first time in human RA, the efficacy of autologous Tregs in reducing the inflammatory activity of synovial tissue cell cultures ex vivo, while in the synovium FoxP3+ Tregs of patients with RA are reduced compared with peripheral blood and synovial fluid. This local imbalance of Th1 and Treg may be responsible for repeated rheumatic flares and thus will be of interest as a target for future treatments.

Topics: Adult; Aged; Aged, 80 and over; Arthritis, Rheumatoid; Biomarkers; CD3 Complex; Cells, Cultured; Female; Forkhead Transcription Factors; Humans; Immunohistochemistry; Interferon-gamma; Interleukin-10; Lymphocyte Count; Male; Middle Aged; Osteoarthritis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Synovial Membrane; T-Lymphocytes, Regulatory; Th1 Cells; Transforming Growth Factor beta

To determine whether an intra-articular injection of recombinant human bone morphogenetic protein-2 (rhBMP-2) alleviates cartilage degradation in a rat model of osteoarthritis (OA) of the lumbar facet joint.. The right-side facet joint OA model was created by an intra-articular injection of collagenase (type II) 2 weeks before treatment. The OA rats were divided into four groups: (1) no treatment, or intra-articular injection of either (2) saline, (3) rhBMP-2 10 ng, or (4) rhBMP-2 100 ng. The left-side facet joint served as the normal control. At 3 and 6 weeks after treatment, histological analyses were performed on the cartilage, synovium, subchondral bone and bone marrow. The cartilage and synovium were graded using a modified Mankin score and a synovium score system. Extracellular type II collagen was evaluated by immunohistochemistry.. Intra-articular injection of collagenase causes OA-like changes in the facet joint. OA rats treated with rhBMP-2 at both dosages tested showed reduced severity of their cartilage lesions compared with untreated and saline-treated groups. There was a statistically significant difference in the modified Mankin score compared to the untreated and saline-treated groups. However, some rhBMP-2-treated rats at the higher dose (100 ng) showed, as a side effect, joint space obliteration caused by cartilage overgrowth. Also OA rats treated with 100 ng of rhBMP-2 displayed a significant synovium reaction at 3 weeks compared with that in other groups. Immunohistochemical analysis showed that treatment with rhBMP-2 significantly increased the content of type II collagen.. This study demonstrates the potential efficacy of rhBMP-2 in the alleviation of arthritic changes in a rat model of OA of the lumbar facet joint. However, treatment with a high dosage of rhBMP-2 caused adverse side effects in some animals.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cartilage, Articular; Collagen Type II; Disease Models, Animal; Dose-Response Relationship, Drug; Matrix Metalloproteinase 8; Osteoarthritis; Random Allocation; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Staining and Labeling; Synovial Membrane; Transforming Growth Factor beta; Zygapophyseal Joint

Osteoarthritis (OA) is characterized by alterations to subchondral bone as well as articular cartilage. Changes to bone in OA have also been identified at sites distal to the affected joint, which include increased bone volume fraction and reduced bone mineralization. Altered bone remodelling has been proposed to underlie these bone changes in OA. To investigate the molecular basis for these changes, we performed microarray gene expression profiling of bone obtained at autopsy from individuals with no evidence of joint disease (control) and from individuals undergoing joint replacement surgery for either degenerative hip OA, or fractured neck of femur (osteoporosis [OP]). The OP sample set was included because an inverse association, with respect to bone density, has been observed between OA and the low bone density disease OP. Compugen human 19K-oligo microarray slides were used to compare the gene expression profiles of OA, control and OP bone samples. Four sets of samples were analyzed, comprising 10 OA-control female, 10 OA-control male, 10 OA-OP female and 9 OP-control female sample pairs. Print tip Lowess normalization and Bayesian statistical analyses were carried out using linear models for microarray analysis, which identified 150 differentially expressed genes in OA bone with t scores above 4. Twenty-five of these genes were then confirmed to be differentially expressed (P < 0.01) by real-time PCR analysis. A substantial number of the top-ranking differentially expressed genes identified in OA bone are known to play roles in osteoblasts, osteocytes and osteoclasts. Many of these genes are targets of either the WNT (wingless MMTV integration) signalling pathway (TWIST1, IBSP, S100A4, MMP25, RUNX2 and CD14) or the transforming growth factor (TGF)-beta/bone morphogenic protein (BMP) signalling pathway (ADAMTS4, ADM, MEPE, GADD45B, COL4A1 and FST). Other differentially expressed genes included WNT (WNT5B, NHERF1, CTNNB1 and PTEN) and TGF-beta/BMP (TGFB1, SMAD3, BMP5 and INHBA) signalling pathway component or modulating genes. In addition a subset of genes involved in osteoclast function (GSN, PTK9, VCAM1, ITGB2, ANXA2, GRN, PDE4A and FOXP1) was identified as being differentially expressed in OA bone between females and males. Altered expression of these sets of genes suggests altered bone remodelling and may in part explain the sex disparity observed in OA.

Topics: Aged; Aged, 80 and over; Bone Morphogenetic Proteins; Bone Remodeling; Female; Gene Expression Profiling; Humans; Male; Microarray Analysis; Middle Aged; Osteoarthritis; Signal Transduction; Transforming Growth Factor beta; Wnt Proteins

Data on synovial fluid (SF) cytokine concentrations in patients with reactive arthritis (ReA) or undifferentiated spondyloarthropathy (uSpA) are limited and contradictory. We measured levels of several proinflammatory and immunoregulatory cytokines in SF and sera from patients with ReA/uSpA.. Interleukin 17 (IL-17), IL-6, interferon-g (IFN-g), and IL-12p40, and immunoregulatory cytokines IL-10 and transforming growth factor-beta (TGF-beta) were assayed using ELISA in SF specimens from 51 patients with ReA/uSpA (ReA 21, uSpA 30), 40 patients with rheumatoid arthritis (RA), and 11 patients with osteoarthritis (OA). IL-17, IL-6, IFN-g, and IL-10 levels were also measured in paired sera samples from patients with ReA/uSpA.. SF concentrations of IL-17, IL-6, TGF-beta, and IFN-g were significantly higher in patients with ReA/uSpA as compared to RA patients (for IL-17 median 46 pg/ml, range < 7.8-220 vs median < 7.8 pg/ml, range < 7.8-136, p < 0.05; for TGF-beta median 4.2 ng/ml, range 1.32-12 vs median 3.01 ng/ml, range 0.6-9.6, p < 0.01; for IL-6 median 58 ng/ml, range 2-540 vs median 34.5 ng/ml, range < 0.009-220, p < 0.05; for IFN-g median 290 pg/ml, range < 9.4-1600 vs median 100 pg/ml, range < 9.4-490, p < 0.05). SF levels of IL-10 were comparable but the ratio of IFN-g/IL-10 was significantly higher in ReA/uSpA patients than RA patients (median 3.18, range 0.06-200 for ReA/uSpA vs median 1.0, range 0.03-26.9 for RA; p < 0.05). IL-17, IL-6, IL-10, and IFN-g SF levels were significantly higher than paired serum levels in ReA/uSpA patients (p < 0.01 for IL-17, p < 0.0001 for IL-6, p < 0.0001 for IL-10, and p < 0.001 for IFN-g).. Increased IL-17, IL-6, TGF-beta, and IFN-g concentrations in ReA/uSpA than in RA suggest that Th1 and Th17 cells could be the major agents in inflammation in ReA/uSpA.

Topics: Adolescent; Adult; Arthritis, Reactive; Arthritis, Rheumatoid; Cytokines; Female; Humans; Interferon-gamma; Interleukin-12 Subunit p40; Interleukin-17; Interleukin-6; Male; Middle Aged; Osteoarthritis; Prohibitins; Spondylarthropathies; Synovial Fluid; Transforming Growth Factor beta

Smad3 deficiency accelerates chondrocyte maturation and leads to osteoarthritis. Primary chondrocytes without Smad3 lack compensatory increases of TGF-beta signaling factors, but BMP-related gene expression is increased. Smad2 or Smad3 overexpression and BMP blockade abrogate accelerated maturation in Smad3-/- chondrocytes. BMP signaling is increased in TGF-beta deficiency and is required for accelerated chondrocyte maturation.. Disruption of TGF-beta signaling results in accelerated chondrocyte maturation and leads to postnatal dwarfism and premature osteoarthritis. The mechanisms involved in this process were studied using in vitro murine chondrocyte cultures.. Primary chondrocytes were isolated from the sterna of neonatal wildtype and Smad3-/- mice. Expressions of maturational markers, as well as genes involved in TGF-beta and BMP signaling were examined. Chondrocytes were treated with TGF-beta and BMP-2, and effects on maturation-related genes and BMP/TGF-beta responsive reporters were examined. Recombinant noggin or retroviral vectors expressing Smad2 or Smad3 were added to the cultures.. Expression of colX and other maturational markers was markedly increased in Smad3-/- chondrocytes. Smad3-/- chondrocytes lacked compensatory increases in Smad2, Smad4, TGFRII, Sno, or Smurf2 and had reduced expression of TGF-beta1 and TGFRI. In contrast, Smad1, Smad5, BMP2, and BMP6 expression was increased, suggesting a shift from TGF-beta toward BMP signaling. In Smad3-/- chondrocytes, alternative TGF-beta signaling pathways remained responsive, as shown by luciferase assays. These non-Smad3-dependent TGF-beta pathways reduced colX expression and alkaline phosphatase activity in TGF-beta-treated Smad3-/- cultures, but only partially. In contrast, Smad3-/- chondrocytes were more responsive to BMP-2 treatment and had increased colX expression, phosphoSmads 1, 5, and 8 levels, and luciferase reporter activity. Overexpression of both Smad2 and Smad3 blocked spontaneous maturation in Smad3-deficient chondrocytes. Maturation was also abrogated by the addition of noggin, an extracellular BMP inhibitor.. These findings show a key role for BMP signaling during the chondrocyte maturation, occurring with loss of TGF-beta signaling with important implications for osteoarthritis and cartilage diseases.

Topics: Animals; Bone Morphogenetic Proteins; Cartilage Diseases; Cell Differentiation; Cells, Cultured; Chondrocytes; Gene Expression Regulation; Mice; Mice, Knockout; Osteoarthritis; Signal Transduction; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta

Osteoarthritis is a common malady of the musculoskeletal system affecting the articular cartilage. The increased frequency of osteoarthritis with aging indicates the complex etiology of this disease, which includes pathophysiology and joint stability including biomechanics. The balance between anabolic morphogens and growth factors and catabolic cytokines is at the crux of the problem of osteoarthritis. One such signal is transforming growth factor-beta (TGF-beta). The impaired TGF-beta signaling has been identified as a culprit in old mice in a recent article in this journal. This commentary places this discovery in the context of anabolic and catabolic signals and articular cartilage homeostasis in the joint.

Topics: Aging; Animals; Cartilage, Articular; Humans; Mice; Osteoarthritis; Signal Transduction; Transforming Growth Factor beta

Articular cartilage degeneration in osteoarthritis (OA) involves type II collagen degradation and chondrocyte differentiation (hypertrophy). Because these changes resemble growth plate remodeling, we hypothesized that collagen degradation may be inhibitable by growth factors known to suppress growth plate hypertrophy, namely transforming growth factor (TGF)-beta2, fibroblast growth factor (FGF)-2, and insulin. Full-depth explants of human OA knee articular cartilage from arthroplasty were cultured with TGF-beta2, FGF-2, and insulin in combination (growth factors) or individually. In cultured explants from five OA patients, collagenase-mediated type II collagen cleavage was significantly down-regulated by combined growth factors as measured by enzyme-linked immunosorbent assay. Individually, FGF-2 and insulin failed to inhibit collagen cleavage in some OA explants whereas TGF-beta2 reduced collagen cleavage in these 5 explants and in 19 additional explants. Moreover, TGF-beta2 effectively suppressed cleavage at low concentrations. Together or individually these growth factors did not inhibit glycosaminoglycan (primarily aggrecan) degradation while TGF-beta2 occasionally did. Semiquantitative reverse transcriptase-polymerase chain reaction of articular cartilage from six OA patients revealed that TGF-beta2 suppressed expression of matrix metalloproteinase-13 and matrix metalloproteinase-9, early (PTHrP) and late (COL10A1) differentiation-related genes, and proinflammatory cytokines (interleukin-1beta, tumor necrosis factor-alpha). In contrast, TGF-beta2 up-regulated PGES-1 expression and prostaglandin E(2) release. These observations show that TGF-beta2 can suppress collagen resorption and chondrocyte differentiation in OA cartilage and that this may be mediated by prostaglandin E(2). Therefore TGF-beta2 could provide therapeutic control of type II collagen degeneration in OA.

Topics: Aged; Aged, 80 and over; Cartilage; Cells, Cultured; Chondrocytes; Collagen; Dinoprostone; Enzyme-Linked Immunosorbent Assay; Female; Fibroblast Growth Factor 2; Gene Expression; Glycosaminoglycans; Humans; Insulin; Male; Organ Culture Techniques; Osteoarthritis; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta; Transforming Growth Factor beta2

The primary feature of osteoarthritis is cartilage loss. In addition, osteophytes can frequently be observed. Transforming growth factor-beta (TGFbeta) has been suggested to be associated with protection against cartilage damage and new cartilage formation as seen in osteophytes.. To study TGFbeta and TGFbeta signalling in experimental osteoarthritis to gain insight into the role of TGFbeta in cartilage degradation and osteophyte formation during osteoarthritis progression.. Histological sections of murine knee joints were stained immunohistochemically for TGFbeta3 and phosphorylated SMAD-2 (SMAD-2P). Expression patterns were studied in two murine osteoarthritis models, representing spontaneous (STR/ort model) and instability-associated osteoarthritis (collagenase-induced instability model).. TGFbeta3 and SMAD-2P staining was increasingly reduced in cartilage during osteoarthritis progression in both models. Severely damaged cartilage was negative for TGFbeta3. In contrast, bone morphogenetic protein-2 (BMP-2) expression was increased. In chondrocyte clusters, preceding osteophyte formation, TGFbeta3 and SMAD-2P were strongly expressed. In early osteophytes, TGFbeta3 was found in the outer fibrous layer, in the peripheral chondroblasts and in the core. Late osteophytes expressed TGFbeta3 only in the fibrous layer. SMAD-2P was found throughout the osteophyte at all stages. In the late-stage osteophytes, BMP-2 was strongly expressed.. Data show that lack of TGFbeta3 is associated with cartilage damage, suggesting loss of the protective effect of TGFbeta3 during osteoarthritis progression. Additionally, our results indicate that TGFbeta3 is involved in early osteophyte development, whereas BMP might be involved in late osteophyte development.

Topics: Animals; Arthritis, Experimental; Cartilage, Articular; Chondrogenesis; Disease Progression; Joint Instability; Male; Mice; Ossification, Heterotopic; Osteoarthritis; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta; Transforming Growth Factor beta3

Transforming growth factor (TGF)-beta1 is a pleiotropic cytokine with many functions, including those related to growth modulation, immunosuppression, and pro-inflammation, in a wide variety of cell types. In this study, we investigated the ability of TGF-beta1 to regulate RANTES production by activated rheumatoid synovial fibroblasts. Fibroblast-like synoviocytes (FLS) were cultured in the presence of TGF-beta1 and IL-1beta, IL-15, TNFalpha, or IL-17, and the secretion of RANTES into culture supernatants was measured by enzyme-linked immunosorbent assay (ELISA). Expression of RANTES encoded mRNA was determined by reverse transcription-polymerase chain reaction (RT-PCR), and NF-kappaB binding activity for RANTES transcription was determined by electrophoretic mobility shift assay (EMSA). We found that the concentrations of RANTES in synovial fluid (SF) from rheumatoid arthritis (RA) patients were lower than in SF from osteoarthritis (OA) patients, whereas the concentrations of TGF-beta1 were higher in RA SF than in OA SF. TGF-beta1 dose-dependently inhibited TNFalpha-induced production of RANTES protein and mRNA from RA FLS. Addition of RA SF with high-level TGF-beta1 mimicked the effect of TGF-beta1 on TNFalpha-induced RANTES production, which was inhibited by treatment with anti-TGF-beta1 neutralizing antibody. TGF-beta1 blocked the degradation of cytosolic IkappaB-alpha and the translocation of activated NF-kappaB to the nucleus. EMSA showed that the inhibitory effect of TGF-beta1 was associated with decreased binding of NF-kappaB to the RANTES promoter. These results suggest that elevated TGF-beta1 in rheumatoid synovial tissue may suppress joint inflammation by inhibiting RANTES secretion from synovial fibroblasts, thus blocking the infiltration of immune cells. These findings may provide an explanation for the mechanism by which TGF-beta1 regulates immune function in RA.

Topics: Adult; Aged; Aged, 80 and over; Arthritis, Rheumatoid; Cells, Cultured; Chemokine CCL5; Down-Regulation; Female; Fibroblasts; Humans; Male; Middle Aged; NF-kappa B; Osteoarthritis; Promoter Regions, Genetic; Protein Binding; RNA, Messenger; Synovial Fluid; Synovial Membrane; Transcription, Genetic; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

Cartilage damage in osteoarthritis (OA) is considered an imbalance between catabolic and anabolic factors, favoring the catabolic side. We assessed whether adenoviral overexpression of transforming growth factor-beta (TGFbeta) enhanced cartilage repair and whether TGFbeta-induced fibrosis was blocked by local expression of the intracellular TGFbeta inhibitor Smad7. We inflicted cartilage damage by injection of interleukin-1 (IL-1) into murine knee joints. After 2 days, we injected an adenovirus encoding TGFbeta. On day 4, we measured proteoglycan (PG) synthesis and content. To examine whether we could block TGFbeta-induced fibrosis and stimulate cartilage repair simultaneously, we injected Ad-TGFbeta and Ad-Smad7. This was performed both after IL-1-induced damage and in a model of primary OA. In addition to PG in cartilage, synovial fibrosis was measured by determining the synovial width and the number of procollagen I-expressing cells. Adenoviral overexpression of TGFbeta restored the IL-1-induced reduction in PG content and increased PG synthesis. TGFbeta-induced an elevation in PG content in cartilage of the OA model. TGFbeta-induced synovial fibrosis was strongly diminished by simultaneous synovial overexpression of Smad7 in the synovial lining. Of great interest, overexpression of Smad7 did not reduce the repair-stimulating effect of TGFbeta on cartilage. Adenoviral overexpression of TGFbeta stimulated repair of IL-1- and OA-damaged cartilage. TGFbeta-induced synovial fibrosis was blocked by locally inhibiting TGFbeta signaling in the synovial lining by simultaneously transfecting it with an adenovirus overexpressing Smad7.

Topics: Animals; Cartilage; Disease Models, Animal; Fibrosis; Genetic Vectors; Interleukin-1; Knee Joint; Mice; Mice, Inbred C57BL; Osteoarthritis; Recombinant Proteins; Smad7 Protein; Transfection; Transforming Growth Factor beta; Wound Healing

To investigate the modulation of expression of proteinase-activated receptor-2 (PAR-2) in articular chondrocytes by inflammatory cytokines.. Articular synovium and cartilage tissues were collected from eight patients with osteoarthritis (OA), and three patients without arthropathy ("normal"). Chondrocytes were stimulated with interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha or transforming growth factor (TGF)-beta1. The expression of PAR-2 was detected using reverse transcriptase-polymerase chain reaction (PCR), Western blotting and immunofluorescence. Quantitative PCR was performed to assess the expression levels of PAR-2 messenger RNA (mRNA).. The expression of PAR-2 mRNA was demonstrated in both OA and normal chondrocytes as well as in synovial fibroblasts. However, the level of PAR-2 in OA chondrocytes was much higher than in normal chondrocytes. Long-term culture revealed that PAR-2 mRNA expression was maintained up to three passages in OA but not in normal chondrocytes. IL-1beta and TNF-alpha both upregulated PAR-2 expression in normal and OA chondrocytes. In contrast, TGF-beta1 significantly decreased expression of PAR-2 in OA chondrocytes but increased PAR-2 in normal chondrocytes.. Overexpression of PAR-2 in OA chondrocytes is upregulated by proinflammatory cytokines IL-1beta and TNF-alpha, and down-regulated by regulatory cytokine TGF-beta1. PAR-2 may be involved in the pathogenesis of OA.

Topics: Aged; Aged, 80 and over; Cartilage, Articular; Cell Line; Cell Membrane; Cells, Cultured; Chondrocytes; Cytokines; Cytoplasm; Dose-Response Relationship, Immunologic; Female; Fibroblasts; Humans; Interleukin-1beta; Male; Middle Aged; Osteoarthritis; Receptor, PAR-2; RNA, Messenger; Synovial Membrane; Time Factors; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Up-Regulation

The purpose of the current study was to investigate the abnormal expression of Col X, PTHrP, TGF-beta, bFGF, and VEGF in cartilage from patients with Kashin-Beck disease (KBD) to understand the pathogenesis of chondronecrosis in KBD. Articular cartilage and growth plate cartilage collected were divided into four groups: control children (8 samples, 5 cases), KBD children (19 samples, 9 cases), control adults (8 samples, 6 cases), and KBD adults (16 samples, 15 cases). The presence of PTHrP, TGF-beta1, bFGF, VEGF, and collagen X in articular cartilage and in growth plate cartilage was analyzed by immunohistochemistry. Articular cartilage and growth plate were each divided in three zones, and the rate of positive cells was counted by light microscope for cytoplasmic and pericellular staining. Results showed that (1) in KBD children, Col X expression was lower in the deep zone of growth plate cartilage than in normal children; in articular cartilage of KBD adults, however, collagen X expression was higher in the middle zone compared to the controls; (2) staining for bFGF, PTHrP, TGF-beta1, and VEGF in KBD adult patients was prominent in the chondrocyte clusters and the eroded surface of articular cartilage, and the percentage of chondrocyte staining was significantly higher than in control samples (t = 3.64-10.34, df = 12 for children and 19 for adults, P = 0.002-0.0001); and (3) the enhanced PTHrP, TGF-beta1, and VEGF staining in the deep and middle zone of KBD articular cartilage correlated with the high incidence of chondronecrosis in the middle zone (48.5% +/- 10.2%) and deep zone (70.6% +/- 27.0%) of adult KBD cartilage. In conclusion, Col X expression was reduced in areas of chondrocyte necrosis in the deep zone of KBD articular cartilage, indicating changes in terminal chondrocyte differentiation. PTHrP, TGF-beta1, and VEGF expression was significantly altered and indicated degenerative changes in KBD cartilage, which initially resemble those occurring in osteoarthritis, but lead eventually to chondronecrosis, an event not observed in osteoarthritis.

Topics: Adult; Cartilage; Child; Chondrocytes; Collagen; Female; Fibroblast Growth Factors; Gene Expression; Humans; Male; Middle Aged; Osteoarthritis; Parathyroid Hormone-Related Protein; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Cartilage is a support tissue with a poor capacity to self-repair. Its cells, chondrocytes, are responsible for synthesizing and renewing the matrix that surrounds them in a constant turnover mechanism. Autologous chondrocyte implantation (ACI) is one of the techniques that promises to be an alternative to common strategies for chondral lesions. To apply this technique, a large amount of cells must be obtained. In our work, we studied the state of cells from different cartilage (young, aged, and osteoarthritic sheep) cultured in monolayer by analyzing their proliferation rate using bromodeoxyuridine and their gene expression profile by RT-PCR. A decrease was found in expression of type II collagen and aggrecan in aged, osteoarthritic, and passaged chondrocytes. Treatment of cells with growth factors aFGF, IGF-I, TGF-beta, and OP-1 improved the proliferation rate in all cells studied and stimulated gene expression of type II collagen, aggrecan, and TGF-beta. Osteoarthritic cells showed a poor response according to matrix gene expression, while young cells responded properly, and aged chondrocytes showed a moderate response. These results suggest that the state of cartilage may affect the behavior of cultured chondrocytes.

Topics: Aggrecans; Aging; Animals; Bromodeoxyuridine; Cell Proliferation; Cells, Cultured; Chondrocytes; Collagen Type II; Disease Models, Animal; Gene Expression; Intercellular Signaling Peptides and Proteins; Osteoarthritis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sheep; Transforming Growth Factor beta

The aim of this study was to demonstrate the induction of chondrogenesis by transforming growth factor (TGF)-beta1 from synovium-derived mesenchymal stem cells in a three-dimensional polyglycolic acid (PGA) scaffold, and to evaluate the effects of insulin-like growth factor (IGF)-I on TGF-beta1-induced chondrogenesis. Adult human synovial membranes were obtained from the knees of patients with osteoarthritis or rheumatoid arthritis. Cells were expanded in monolayers, seeded onto a PGA scaffold, and cultured for 4 or 8 weeks in chondrogenic medium containing TGF-beta1 with or without IGF-I. As a control, the cells were cultured in chondrogenic medium without TGF-beta1. The glycosaminoglycan content was quantified using dimethylmethylene blue dye-binding assay, and the DNA content was measured fluorometrically. Histological examination was also performed using safranin-O staining. The expression of mRNA for aggrecan and collagen type II was confirmed by RT-PCR. After 4 weeks of cultivation with TGF-beta1, the cells differentiated to a chondrocytic phenotype, and these chondrogeneses were more potent when cultured for 8 weeks. The combination of IGF-I and TGF-beta1 produced higher amounts of glycosaminoglycan than TGF-beta1 alone at 8 weeks. In conclusions, chondrogenesis from human synovium-derived mesenchymal cells was identified, and IGF-I plays a role in maintaining the extracellular matrix in combination with TGF-beta1.

Topics: Adult; Aged; Aged, 80 and over; Arthritis, Rheumatoid; Cells, Cultured; Chondrogenesis; DNA; Glycosaminoglycans; Humans; Insulin-Like Growth Factor I; Knee Joint; Mesenchymal Stem Cells; Middle Aged; Osteoarthritis; Polyglycolic Acid; Synovial Membrane; Transforming Growth Factor beta

To evaluate the expression and function of the orphan nuclear receptor Rev-ErbAalpha in articular cartilage and to investigate its role in osteoarthritis (OA).. Expression of Rev-ErbAalpha was analyzed at both the messenger RNA and protein levels in human and bovine articular cartilage and chondrocytes by real-time polymerase chain reaction (TaqMan) and immunocytochemical techniques. The effects of cartilage catabolic and anabolic agents on the expression of Rev-ErbAalpha were evaluated by TaqMan analysis. Overexpression was achieved by either adenoviral transduction or treatment with a peroxisome proliferator-activated receptor alpha agonist, whereas expression was suppressed by antisense oligonucleotides.. Among the 48 known nuclear receptors, Rev-ErbAalpha was found to be the most highly expressed in OA cartilage. It is known to function as a transcription repressor. Treatment of articular chondrocytes with known catabolic agents resulted in the induction of Rev-ErbAalpha, whereas stimulation with anabolic agents led to a decrease in expression. Overexpression of the nuclear receptor was associated with an increase in the expression of matrix-degrading enzymes such as matrix metalloproteinase 13 and aggrecanase. In contrast, a decrease in Rev-ErbAalpha expression led to a concomitant reduction in the activity of matrix-degrading enzymes.. This study is the first to demonstrate that Rev-ErbAalpha is highly expressed in OA articular chondrocytes and that its expression is modulated by known cartilage catabolic and anabolic stimuli. We also demonstrated that modulation of Rev-ErbAalpha expression in chondrocytes may be a novel means of regulating the expression and production of multiple matrix-degrading enzymes. These observations suggest that Rev-ErbAalpha may be a novel therapeutic target for OA.

Topics: ADAM Proteins; ADAMTS5 Protein; Animals; Antineoplastic Agents; Cartilage; Cattle; Cells, Cultured; Chondrocytes; DNA-Binding Proteins; Down-Regulation; Extracellular Matrix Proteins; Gene Expression; Humans; Insulin-Like Growth Factor I; Interleukin-1; Matrix Metalloproteinase 13; Nuclear Receptor Subfamily 1, Group D, Member 1; Oligonucleotides, Antisense; Osteoarthritis; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Transforming Growth Factor beta; Tretinoin; Tumor Necrosis Factor-alpha; Up-Regulation

Osteoarthritis is the most common form of human arthritis. We investigated the potential role of asporin, an extracellular matrix component expressed abundantly in the articular cartilage of individuals with osteoarthritis, in the pathogenesis of osteoarthritis. Here we report a significant association between a polymorphism in the aspartic acid (D) repeat of the gene encoding asporin (ASPN) and osteoarthritis. In two independent populations of individuals with knee osteoarthritis, the D14 allele of ASPN is over-represented relative to the common D13 allele, and its frequency increases with disease severity. The D14 allele is also over-represented in individuals with hip osteoarthritis. Asporin suppresses TGF-beta-mediated expression of the genes aggrecan (AGC1) and type II collagen (COL2A1) and reduced proteoglycan accumulation in an in vitro model of chondrogenesis. The effect on TGF-beta activity is allele-specific, with the D14 allele resulting in greater inhibition than other alleles. In vitro binding assays showed a direct interaction between asporin and TGF-beta. Taken together, these findings provide another functional link between extracellular matrix proteins, TGF-beta activity and disease, suggesting new therapeutic strategies for osteoarthritis.

Topics: Aggrecans; Aspartic Acid; Carrier Proteins; Chondrogenesis; Chromosome Mapping; Disease Susceptibility; Extracellular Matrix Proteins; Glycoproteins; Humans; In Vitro Techniques; Lectins, C-Type; Minisatellite Repeats; Molecular Sequence Data; Osteoarthritis; Osteoarthritis, Hip; Osteoarthritis, Knee; Polymorphism, Genetic; Proteoglycans; Transforming Growth Factor beta

Osteoarthritis is the most common degenerative disorder of the modern world. However, many basic cellular features and molecular processes of the disease are poorly understood. In the present study we used oligonucleotide-based microarray analysis of genes of known or assumed relevance to the cellular phenotype to screen for relevant differences in gene expression between normal and osteoarthritic chondrocytes. Custom made oligonucleotide DNA arrays were used to screen for differentially expressed genes in normal (n = 9) and osteoarthritic (n = 10) cartilage samples. Real-time polymerase chain reaction (PCR) with gene-specific primers was used for quantification. Primary human adult articular chondrocytes and chondrosarcoma cell line HCS-2/8 were used to study changes in gene expression levels after stimulation with interleukin-1beta and bone morphogenetic protein, as well as the dependence on cell differentiation. In situ hybridization with a gene-specific probe was applied to detect mRNA expression levels in fetal growth plate cartilage. Overall, more than 200 significantly regulated genes were detected between normal and osteoarthritic cartilage (P < 0.01). One of the significantly repressed genes, Tob1, encodes a protein belonging to a family involved in silencing cells in terms of proliferation and functional activity. The repression of Tob1 was confirmed by quantitative PCR and correlated to markers of chondrocyte activity and proliferation in vivo. Tob1 expression was also detected at a decreased level in isolated chondrocytes and in the chondrosarcoma cell line HCS-2/8. Again, in these cells it was negatively correlated with proliferative activity and positively with cellular differentiation. Altogether, the downregulation of the expression of Tob1 in osteoarthritic chondrocytes might be an important aspect of the cellular processes taking place during osteoarthritic cartilage degeneration. Activation, the reinitiation of proliferative activity and the loss of a stable phenotype are three major changes in osteoarthritic chondrocytes that are highly significantly correlated with the repression of Tob1 expression.

Topics: Adult; Aged; Aged, 80 and over; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Cartilage, Articular; Cell Differentiation; Cell Division; Cell Line, Tumor; Cells, Cultured; Chondrocytes; Chondrosarcoma; Female; Gene Expression Profiling; Gene Expression Regulation; Humans; In Situ Hybridization; Interleukin-1; Intracellular Signaling Peptides and Proteins; Ki-67 Antigen; Male; Middle Aged; Oligonucleotide Array Sequence Analysis; Osteoarthritis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta; Tumor Suppressor Proteins

Topics: Carrier Proteins; Case-Control Studies; Extracellular Matrix Proteins; Glycoproteins; Humans; Oligonucleotide Array Sequence Analysis; Osteoarthritis; Polymorphism, Genetic; Risk Factors; RNA, Messenger; Transforming Growth Factor beta

This study attempted to elucidate the genetic and environmental factors influencing interindividual variation of circulating TIMP-1, TIMP-2 and TGF-beta1 and to clarify the relationship between the latter biochemical indices and hand osteoarthritis in an ethnically homogeneous sample. Plasma levels of each of the above biochemical indices were measured in 401 healthy individuals (aged 18-75 years) belonging to 90 nuclear and more complex families. Variance component analysis showed that a major part of the interindividual differences in TGF-beta1, TIMP-1 and TIMP-2 levels was credibly attributable to genetic and familial factors. Adjusted for significant covariates, the putative genetic effects on the above three amounted to 0.40 +/- 0.10, 0.47 +/- 0.11 and 0.72 +/- 0.10, respectively. Common environmental factors, shared by members of the same household, also contributed significantly ( P < 0.01) to variation of each of the biochemical indices and explained between 27.6% (TIMP-2) and 38.7% (TGF-beta1) of their variation. A bivariate analysis revealed a strong and highly significant correlation between TIMP-1 and TGF-beta1 (r = 0.58, P < 0.001), which was due to common genetic and environmental sources (r(G) = 0.62 +/- 0.09, r(E) = 0.31 +/- 0.11, both P < 0.001). The analysis also detected modest but significant genetic correlation between TIMP-1 and TIMP-2 (r(G) = -0.307 +/- 0.108, P < 0.01). The present study evinces a strong genetic dependence for the plasma levels of both TIMPs and TGF-beta1 and provides a basis for the further analysis of genetic variation affecting and regulating the circulatory concentrations of TIMPs and TGF-beta1 in healthy humans.

Topics: Adolescent; Adult; Aged; Female; Genetic Variation; Hand; Humans; Male; Middle Aged; Multivariate Analysis; Nuclear Family; Osteoarthritis; Pedigree; Radiography; Rural Population; Russia; Tissue Inhibitor of Metalloproteinase-1; Tissue Inhibitor of Metalloproteinase-2; Tissue Inhibitor of Metalloproteinases; Transforming Growth Factor beta

To investigate in vivo and in vitro whether macrophages have an intermediate role in transforming growth factor beta (TGFbeta)-induced osteophyte formation.. In vivo, synovial lining macrophages were selectively depleted by injection of clodronate-laden liposomes 7 days prior to injection of 20 ng or 200 ng of TGFbeta into murine knee joints 3 times, on alternate days. Total knee joint sections were obtained on day 7 after the last injection and stained with Safranin O. Production of bone morphogenetic protein 2 (BMP-2) and BMP-4 was determined by immunolocalization. The interaction between murine macrophages and mesenchymal cells (precursors with chondrogenic potential) was studied in vitro using a Transwell system in which RAW macrophages were cocultured with C3H10T1/2 mesenchymal cells. Spheroid neocartilage formation was quantified microscopically after staining with May-Grünwald-Giemsa.. Triple injections of 20 ng or 200 ng of TGFbeta into normal murine knee joints induced significant osteophyte formation at the lateral and medial sites of the patella and femur on day 7 after the last injection. Strikingly, removal of synovial lining macrophages prior to TGFbeta injection resulted in a drastic reduction of osteophyte formation (by 70% and 64% after injection of 20 ng and 200 ng of TGFbeta, respectively). Synovial lining cells produced BMP-2 and BMP-4 after TGFbeta stimulation, whereas BMP-2 and BMP-4 were absent in the synovial tissue after macrophage depletion. In vitro, clustering and spheroid formation of C3H10T1/2 was induced by TGFbeta concentrations of >1 ng/ml. However, in the Transwell system, in the presence of murine macrophages, 0.5 ng/ml of TGFbeta was very effective in generating large spheroids, suggestive of macrophage-derived (co)factors. In coculture supernatants, TGFbeta concentrations were not elevated in the presence of macrophages, indicating generation of other growth factors involved in spheroid formation.. These findings indicate that macrophages are crucial intermediate factors in osteophyte formation induced by TGFbeta, probably by inducing other chondrogenic signals.

Topics: Animals; Antimetabolites; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Bone Morphogenetic Proteins; Cells, Cultured; Chondrocytes; Clodronic Acid; Liposomes; Macrophages; Mesoderm; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Osteoarthritis; Periosteum; Stem Cells; Synovial Membrane; Transforming Growth Factor beta

Aggrecan is degraded by Aggrecanases (ADAMTS-4 and -5) and MMPs, which cleave its core protein at different sites. Transforming growth factor (TGF)beta is known to stimulate matrix formation in cartilage, and ADAMTS-4 production in synoviocytes. The aim of this in-vitro study was to examine the effects of TGFbeta on aggrecanase production in human cartilage.. Expression of ADAMTS-4 and -5 in chondrocyte cultures from normal or osteoarthritic cartilage was studied at mRNA level by RT-PCR. Aggrecanase activity was examined by western blot of aggrecanase-generated neoepitope NITEGE, and by measure of proteoglycan degradation in cartilage explants.. TGFbeta strongly increased mRNA levels of ADAMTS-4, while ADAMTS-5 was expressed in a constitutive way in chondrocytes from normal and osteoathritic cartilage. TGFbeta also increased NITEGE levels and proteoglycan degradation. Addition of an aggrecanase inhibitor blocked the increase of NITEGE, and partially inhibited proteoglycan degradation.. TGFbeta stimulates ADAMTS-4 expression and aggrecan degradation in cartilage. This catabolic action seems to be partially mediated by aggrecanases. It is, therefore, proposed that the role of TGFbeta in cartilage matrix turnover is not limited to anabolic and anti-catabolic actions, but also extends to selective degradation of matrix components such as aggrecan.

Topics: ADAM Proteins; ADAMTS4 Protein; Adolescent; Adult; Aged; Cartilage, Articular; Cells, Cultured; Chondrocytes; Endopeptidases; Female; Humans; Interleukin-1; Male; Matrix Metalloproteinases; Metalloendopeptidases; Middle Aged; Osteoarthritis; Procollagen N-Endopeptidase; Protein Denaturation; Proteoglycans; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

To examine the capacity of recombinant osteogenic protein-1 (OP-1) to inhibit the cartilage extracellular matrix damage that follows treatment with hyaluronan hexasaccharides (HA6).. The effects of OP-1 were examined on isolated human chondrocytes grown in alginate beads as well as articular cartilage slices treated with hyaluronan hexasaccharides. Changes in the relative expression of messenger RNA for hyaluronan synthase- 2, aggrecan and CD44 were determined by competitive quantitative reverse transcriptase-polymerase chain reaction. Cartilage proteoglycan biosynthesis was examined by a (35)S-sulfate incorporation assay. Cell-associated matrix of human chondrocytes was visualized by the use of particle exclusion assay, and alcian blue staining. Cartilage slices were examined for accumulation of proteoglycan by Safranin-O, and hyaluronan by a specific biotinylated probe.. Combined OP-1 and HA6 treatment resulted in enhanced expression of mRNA for aggrecan and HAS-2, compared to the treatment with HA6 only. This increased expression of aggrecan mRNA was paralleled by an increased synthesis of cartilage proteoglycan especially retained in the cell-associated matrix. Co-treatment with OP-1 inhibited the HA6-induced depletion of cell-associated matrices as well as HA6-induced depletion of hyaluronan and proteoglycan within cartilage tissue slices.. These results demonstrate that OP-1 can abrogate the catabolic events associated with a HA6-induced matrix depletion model of osteoarthritis. The mRNA levels of two major cartilage extracellular matrix components, aggrecan and hyaluronan synthase-2 are enhanced above values obtained by either OP-1 or HA6 treatments alone.

Topics: Activin Receptors, Type I; Adult; Aged; Aggrecans; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Cartilage, Articular; Cells, Cultured; Chondrocytes; Extracellular Matrix; Extracellular Matrix Proteins; Glucuronosyltransferase; Humans; Hyaluronan Receptors; Hyaluronan Synthases; Hyaluronic Acid; Lectins, C-Type; Middle Aged; Osteoarthritis; Proteins; Proteoglycans; RNA; Transferases; Transforming Growth Factor beta

To compare gene expression in normal and osteoarthritic (OA) human chondrocytes using microarray technology. Of the novel genes identified, we selected follistatin, a bone morphogenetic protein (BMP) antagonist, and investigated its expression/regulation as well as that of 3 other antagonists, gremlin, chordin, and noggin, in normal and OA chondrocytes and synovial fibroblasts.. Basal and induced gene expression were determined using real-time polymerase chain reaction. Gene regulation was monitored following treatment with inflammatory, antiinflammatory, growth, and developmental factors. Follistatin protein production was measured using a specific enzyme-linked immunosorbent assay, and localization of follistatin and gremlin in cartilage was determined by immunohistochemical analysis.. All BMP antagonists except noggin were expressed in chondrocytes and synovial fibroblasts. Follistatin and gremlin were significantly up-regulated in OA chondrocytes but not in OA synovial fibroblasts. Chordin was weakly expressed in normal and OA cells. Production of follistatin protein paralleled the gene expression pattern. Follistatin and gremlin were expressed preferentially by the chondrocytes at the superficial layers of cartilage. Tumor necrosis factor alpha and interferon-gamma significantly stimulated follistatin expression but down-regulated expression of gremlin. Interleukin-1beta (IL-1beta) had no effect on follistatin but reduced gremlin expression. Conversely, BMP-2 and BMP-4 significantly stimulated expression of gremlin but down-regulated that of follistatin. IL-13, dexamethasone, transforming growth factor beta1, basic fibroblast growth factor, platelet-derived growth factor type BB, and endothelial cell growth factor down-regulated the expression of both antagonists.. This study is the first to show the possible involvement of follistatin and gremlin in OA pathophysiology. The increased activin/BMP-binding activities of these antagonists could affect tissue remodeling. The data suggest that follistatin and gremlin might appear at different stages during the OA process, making them interesting targets for the treatment of this disease.

Topics: Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Bone Morphogenetic Proteins; Carrier Proteins; Cells, Cultured; Chondrocytes; Down-Regulation; Enzyme-Linked Immunosorbent Assay; Fibroblasts; Follistatin; Gene Expression; Gene Expression Regulation; Glycoproteins; Humans; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Interferon-gamma; Interleukin-1; Osteoarthritis; Protein Array Analysis; Proteins; Synovial Membrane; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Up-Regulation

Bone morphogenetic protein (BMP) and transforming growth factor beta (TGFbeta) are potent anabolic factors in adult articular chondrocytes. In this study, we investigated whether intracellular inhibitors of BMP and TGFbeta signaling, inhibitory Smad6 (I-Smad6) and I-Smad7, are expressed in articular chondrocytes in normal and osteoarthritic (OA) cartilage, and whether their expression shows a correlation with the anabolic activity of OA chondrocytes in vivo and after interleukin-1beta (IL-1beta) stimulation in vitro.. RNA isolated directly from normal and OA human knee cartilage as well as from cultured articular chondrocytes was analyzed by (quantitative) polymerase chain reaction technology. Immunolocalization of the I-Smads was performed on tissue sections and compared with the anabolic cellular activity as documented by in situ hybridization experiments for aggrecan and type II collagen.. Both Smad6 and Smad7 were expressed in all samples of normal and OA cartilage. Immunostaining (including confocal microscopy) confirmed the presence of Smad6 and Smad7 in the majority of normal and degenerated articular chondrocytes; localization was mostly cytoplasmic. No correlation between expression of the main anabolic genes and expression of the I-Smads was found. In cultured articular chondrocytes, stimulation with IL-1beta showed up-regulation of Smad7, whereas Smad6 was down-regulated.. Both Smad6 and Smad7 are expressed in adult human articular chondrocytes. The primarily cytoplasmic localization suggests permanent activation of the I-Smads in articular cartilage in vivo. No evidence was found that up-regulation or down-regulation of I-Smads in OA cartilage correlates directly with the anabolic (or catabolic) activity of articular chondrocytes. The regulation in chondrocytes of Smad6 and Smad7 expression by IL-1beta suggests a potentially important role of IL-1beta signaling in chondrocytes, via indirect influencing of the BMP/TGFbeta signaling cascade.

Topics: Adult; Aged; Aged, 80 and over; Aggrecans; Bone Morphogenetic Proteins; Cartilage, Articular; Case-Control Studies; Cells, Cultured; Chondrocytes; Collagen Type II; DNA-Binding Proteins; Extracellular Matrix Proteins; Humans; Immunohistochemistry; Interleukin-1; Lectins, C-Type; Microscopy, Confocal; Middle Aged; Osteoarthritis; Proteoglycans; RNA, Messenger; Smad6 Protein; Smad7 Protein; Subcellular Fractions; Trans-Activators; Transforming Growth Factor beta

This study compares the regulation of three isoforms of hyaluronan synthase (HAS1, HAS2, and HAS3) transcripts and hyaluronan (HA) production by cytokines in human synovial fibroblastic cells derived from tissue from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Levels of HAS mRNA of the cells with or without stimulation were detected using a real-time fluorescence polymerase chain reaction detection system. Concentrations of HA in the culture supernatants of the cells were measured by a sandwich binding protein assay. Molecular weight of HA was evaluated by agarose gel electrophoresis. The relative proportions of the expression pattern of HAS isoforms was similar between RA and OA tissue-derived cells. HAS1 mRNA was upregulated by transforming growth factor-beta and HAS3 mRNA was upregulated by interleukin-1beta and somewhat by tumor necrosis factor-alpha in the RA cells. HAS2 remained unchanged. Differences in the expression pattern of HAS1, HAS2, and HAS3 mRNA by cytokines suggest that these three isoforms are independently and differentially regulated, and each isoform of HAS may have a different role in arthritic joint disease.

Topics: Adult; Arthritis, Rheumatoid; Cells, Cultured; Cytokines; Female; Gene Expression Regulation, Enzymologic; Glucuronosyltransferase; Humans; Hyaluronan Synthases; Hyaluronic Acid; Interleukin-1; Isoenzymes; Joint Capsule; Middle Aged; Osteoarthritis; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Topics: Calcium Pyrophosphate; Chondrocalcinosis; Crystallization; Humans; Osteoarthritis; Synovial Fluid; Transforming Growth Factor beta

Small proteoglycans (PGs) may accumulate in late stage osteoarthritis even as aggrecan is lost. It is not clear what role transforming growth factor (TGF) beta has in this accumulation. Our goal was to investigate the ability of TGF beta 1 to modulate the synthesis and accumulation of decorin, biglycan, and fibromodulin in cartilage explants cultured under conditions in which aggrecan synthesis remains relatively constant.. Articular cartilage was cultured in the presence or absence of 4 ng/ml TGF beta 1 for up to 16 days. Material extracted from cartilage was assayed for 35SO(4)-large and small PGs and for total endogenous decorin, biglycan and fibromodulin.. The synthesis of 35SO(4)-small PGs increased during the 16 days in culture in response to TGF beta 1, but declined in control cultures. The difference in 35SO(4)-decorin between TGF beta 1 and control samples reached nine-fold after 16 days, while the difference in total endogenous decorin was less than 1.5-fold. 35SO(4)-decorin, which was present in TGF beta 1-treated cultures had an identical core protein, but a longer glycosaminoglycan chain than that of decorin in control cultures. No significant differences in endogenous biglycan were detected, but accumulation of fibromodulin in TGF beta 1 explants exceeded fibromodulin in controls, on average, by 3.8-fold. Fibromodulin was present in cartilage in both keratan sulfate- and non-sulfated oligosaccharide-substituted forms.. The accumulation of each of the three small PGs was affected to a different extent in response to TGF beta 1. Of the three, fibromodulin content was most rapidly augmented in response to TGF beta 1.

Topics: Animals; Autoradiography; Biglycan; Blotting, Western; Carrier Proteins; Cartilage, Articular; Cells, Cultured; Decorin; Dogs; Electrophoresis, Polyacrylamide Gel; Extracellular Matrix Proteins; Fibromodulin; Glycosylation; Luminescent Measurements; Osteoarthritis; Proteoglycans; Transforming Growth Factor beta

Bone- and cartilage-derived morphogenetic proteins (BMPs and CDMPs), which are TGFbeta superfamily members, are growth and differentiation factors that have been recently isolated, cloned and biologically characterized. They are important regulators of key events in the processes of bone formation during embryogenesis, postnatal growth, remodelling and regeneration of the skeleton. In the present study, we used immunohistochemical methods to investigate the distribution of BMP-2, -3, -5, -6, -7 and CDMP-1, -2, -3 in human osteophytes (abnormal bony outgrowths) isolated from osteoarthritic hip and knee joints from patients undergoing total joint replacement surgery. All osteophytes consisted of three different areas of active bone formation: (1) endochondral bone formation within cartilage residues; (2) intramembranous bone formation within the fibrous tissue cover and (3) bone formation within bone marrow spaces. The immunohistochemistry of certain BMPs and CDMPs in each of these three different bone formation sites was determined. The results indicate that each BMP has a distinct pattern of distribution. Immunoreactivity for BMP-2 was observed in fibrous tissue matrix as well as in osteoblasts; BMP-3 was mainly present in osteoblasts; BMP-6 was restricted to young osteocytes and bone matrix; BMP-7 was observed in hypertrophic chondrocytes, osteoblasts and young osteocytes of both endochondral and intramembranous bone formation sites. CDMP-1, -2 and -3 were strongly expressed in all cartilage cells. Surprisingly, BMP-3 and -6 were found in osteoclasts at the sites of bone resorption. Since a similar distribution pattern of bone morphogenetic proteins was observed during embryonal bone development, it is suggested that osteophyte formation is regulated by the same molecular mechanism as normal bone during embryogenesis.

Topics: Aged; Aged, 80 and over; Biomarkers; Bone and Bones; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 3; Bone Morphogenetic Protein 5; Bone Morphogenetic Protein 6; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Bone Regeneration; Femur Head; Growth Differentiation Factor 5; Humans; Immunohistochemistry; Middle Aged; Osteoarthritis; Osteoarthritis, Hip; Osteoarthritis, Knee; Tibia; Transforming Growth Factor beta

To determine the level of anabolic response when chondrocytes isolated from human osteoarthritic cartilage are stimulated with 2 doses of insulin-like growth factor-I (IGF-I) for extended culture periods.. Human chondrocytes were isolated from knee cartilage removed at the time of joint replacement surgery for osteoarthritis (OA). The cells were cultured in alginate beads under serum-free conditions and treated with 100 ng/ml or 1000 ng/ml of human recombinant IGF-I. Response was measured during culture periods of 1 to 28 days by determining the level of radiolabeled sulfate incorporated into alcian blue precipitable material and by measuring the level of total proteoglycan accumulation using the dimethylmethylene blue (DMB) assay. For the latter assay, cultures treated with osteogenic protein-1 (OP-1) were used for comparison to IGF-I. Results were normalized to cell numbers using DNA measurements.. The level of IGF-I stimulated sulfate incorporation relative to untreated controls increased with time in culture, with a peak response occurring between days 7 and 14 of culture. There was no significant difference between the 2 IGF-I doses. Despite the stimulation of sulfate incorporation, the DMB assay did not reveal a significant accumulation of proteoglycans in the cell-associated and further-removed matrix with either dose of IGF-I in cultures carried out to 21 days. In contrast, compared to controls, OP-1 at 100 ng/ml stimulated a 3-fold increase in matrix proteoglycan at day 21 of culture.. Prolonged IGF-I treatment of human OA chondrocytes in serum-free alginate cultures stimulated sulfate incorporation without significant accumulation of a proteoglycan matrix in longterm cultures. However, significant proteoglycan accumulation was seen in cultures treated with OP-1, suggesting it is a better stimulator of proteoglycan production by OA chondrocytes.

Topics: Aged; Aged, 80 and over; Alginates; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Cartilage, Articular; Cells, Cultured; Chondrocytes; Dose-Response Relationship, Drug; Extracellular Matrix; Glucuronic Acid; Hexuronic Acids; Humans; Intercellular Signaling Peptides and Proteins; Knee Joint; Middle Aged; Osteoarthritis; Proteoglycans; Recombinant Proteins; Time Factors; Transforming Growth Factor beta

Although growth factor therapy could be an attractive method for stimulating the repair of damaged cartilage matrix, there is evidence that with aging and/or with the development of osteoarthritis (OA), articular chondrocytes may become unresponsive to growth factor stimulation. The aim of the current study was to compare the ability of insulin-like growth factor+(IGF-1) and osteogenic protein+(OP-1), alone and in combination, to stimulate human normal and OA chondrocytes in culture.. Chondrocytes isolated by enzymatic digestion of cartilage obtained from subjects undergoing knee replacement for OA (n = 6) or from normal ankle joints of tissue donors (n = 7) were cultured in alginate beads in serum-free medium and treated for 21 days with 100 ng/ml IGF-1, 100 ng/ml OP-1, or both. Controls were treated with vehicle alone. The cultures were evaluated for cell survival, cell number by DNA analysis, matrix production by particle exclusion assay, and level of accumulated proteoglycan by dimethylmethylene blue assay.. After 21 days in serum-free alginate culture, survival of cells from OA cartilage was 65 +/- 2% (mean +/- SEM), while survival of cells from normal cartilage was significantly greater (82 +/- 3%). Treatment with either IGF-1 or OP-1 alone minimally improved survival, while the combination IGF +OP significantly improved survival, to 87 +/- 2% for OA cells and 95+/-1% for normal cells. Cell proliferation was noted only in the IGF+OP group; this was significant for both normal and OA cells ( approximately 2-fold increase in DNA levels). Matrix production, assessed by particle exclusion and by proteoglycan accumulation, was greatest in the cells treated with IGF + OP in both normal and OA cultures. When proteoglycan levels were corrected for cell numbers (mg proteoglycan/ng DNA), a significant increase over control was noted with OP-1 alone and IGF IGF-1 alone, in both normal and OA cultures, with the greatest levels in the combination group (3-fold increase over control).. OP-1 was more potent than IGF-1 in stimulating proteoglycan production in both normal and OA cells. However, the best results were obtained with the combination, suggesting that combined therapy with IGF-1 and OP-1 may be an effective strategy for treating OA cartilage damage.

Topics: Adult; Aged; Alginates; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Cartilage, Articular; Cell Culture Techniques; Cell Differentiation; Cell Division; Cell Survival; Cells, Cultured; Chondrocytes; DNA; Extracellular Matrix; Glucuronic Acid; Hexuronic Acids; Humans; Insulin-Like Growth Factor I; Middle Aged; Osteoarthritis; Proteoglycans; Transforming Growth Factor beta

A synchronized balance between synthesis and breakdown of extracellular matrix (ECM) molecules in normal articular cartilage is disturbed in osteoarthritis (OA). The focus of our study is the anabolic factor, osteogenic protein-1 (OP-1) that is expressed in articular cartilage and is able to induce the synthesis of ECM components. The major aim was to investigate both qualitatively and quantitatively endogenous OP-1 in normal, degenerative, and OA cartilage. Normal and degenerative cartilage was obtained at autopsies from femoral condyles of human organ donors with no documented history of joint disease; OA cartilage was obtained from patients undergoing joint arthroplasty. Appearance of donor cartilage was evaluated by Collins scale, where normal cartilage is assigned grades 0-1, and degenerated cartilage is assigned grades 2-4. OP-1 mRNA expression was assessed by RT-PCR; OP-1 protein (pro- and active forms) was qualitatively analyzed by Western blotting and quantified by OP-1 ELISA. The highest levels of OP-1 expression (mRNA and protein) were detected in normal cartilage of grade 0. The concentration of OP-1 protein was about 50 ng per gram cartilage dry weight. With the progression of cartilage degeneration (increased Collins grades and OA) OP-1 protein was down-regulated up to 9-fold. These changes affected primarily the active form of OP-1. OP-1 message also declined in cartilages with the increase of degenerative changes. In conclusion, an overall decrease in endogenous OP-1 in degenerated and OA tissue suggests that OP-1 could be one of the factors responsible for normal homeostasis and matrix integrity in cartilage.

Topics: Adult; Aged; Aged, 80 and over; Blotting, Western; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Cartilage, Articular; Enzyme-Linked Immunosorbent Assay; Female; Humans; Male; Middle Aged; Osteoarthritis; Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta

Destruction of cartilage in osteoarthritis is a direct effect of an imbalance between catabolic and anabolic activities in the tissue. While a great deal is known about catabolism, we sought to determine the biochemical basis of the anabolic activity.. Cartilage was isolated from normal and osteoarthritic patients and subjected to both cell and explant culture. mRNA expression levels of the growth and differentiation factors bone morphogenetic protein-2 (BMP-2), BMP-4, BMP-6, cartilage-derived morphogenetic protein-1 (CDMP-1), connective tissue growth factor (CTGF), and activin were determined. BMP-2 was localized in osteoarthritic cartilage by immunohistochemistry. To determine the mechanism of BMP-2 stimulation, chondrocytes were cultured with TGF-beta (transforming growth factor-beta), insulin-like growth factor-1 (IGF-1), interleukin-1beta (IL-1beta), and tumor necrosis factor-alpha (TNF-alpha). The BMP-2 response was monitored by quantitative real-time polymerase chain reaction to ascertain mRNA levels and by Western blot analysis, BMP-2 protein quantitation, and immunohistochemistry to determine protein levels.. BMP-2 was found to be up-regulated in osteoarthritic chondrocytes and cartilage. In cell culture, IL-1beta and TNF-alpha increased BMP-2 mRNA and protein levels by eightfold and fifteenfold, respectively, whereas IGF-1 and TGF-beta1 had no effect. In cartilage explant cultures, IL-1beta and TNF-alpha increased BMP-2 levels both intracellularly and extracellularly. Functional relevance was suggested by co-localization of BMP-2 and newly synthesized type-II procollagen within the same cells.. BMP-2 acts as a stimulus of anabolic activities in normal and osteoarthritic chondrocytes. Furthermore, the pro-inflammatory cytokines IL-1beta and TNF-alpha, known to be present in synovium and cartilage of patients with osteoarthritis, stimulate the production of active BMP-2.

Topics: Aged; Aged, 80 and over; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cartilage, Articular; Cell Differentiation; Cells, Cultured; Chondrocytes; Female; Gene Expression Regulation; Humans; Interleukin-1; Male; Middle Aged; Osteoarthritis; RNA, Messenger; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Up-Regulation

To study the effect of interleukin-1 (IL-1 beta) on the metabolism of osteoarthritic and normal mature condylar chondrocytes in temporomandibular joints, and investigate the role of IL-1 beta in the pathogenesis of temporomandibular joint osteoarthritis.. The primary generation of osteoarthritic and normal condylar chondrocytes cultured in the monolayer condition was treated with 20 micrograms.L-1 recombined human interleukin-1 beta (rhIL-1 beta), and then collected to be detected with RT-PCR method for the cellular metabolism including mRNA expression of type II collagen, aggrecan, collagenase, insulin-like growth factor 1 (IGF-1), and transforming growth factor beta 1 (TGF beta 1).. The normal mature condylar chondrocytes showed the obviously decreased mRNA expression of type II collagen and aggrecan after the intervention of exogenous IL-1 beta, but less influence could be found for the collagenase expression. The osteoarthritic condylar chondrocytes exhibited the decreased mRNA expression of type II collagen and collagenase under the effect of IL-1 beta, while the cellular mRNA expression of aggrecan didn't change obviously. The intervention of exogenous IL-1 beta didn't show an obvious influence on the cellular expression of endogenous growth factors such as IGF-1 or TGF-beta 1 for both the normal and osteoarthritic condylar chondrocytes cultured in vitro.. IL-1 beta could not only disturb the expression of cartilage matrix molecules by the normal condylar chondrocytes, which lead to the lesion of condylar cartilage, but also worsen the abnormal cartilage matrix environment within the osteoarthritic condylar cartilage.

Topics: Animals; Cartilage, Articular; Cells, Cultured; Chondrocytes; Collagen Type II; Humans; Interleukin-1beta; Osteoarthritis; Rabbits; Temporomandibular Joint; Transforming Growth Factor beta

To evaluate the contribution of leptin (an adipose tissue-derived hormone) to the pathophysiology of osteoarthritis (OA), by determining the level of leptin in both synovial fluid (SF) and cartilage specimens obtained from human joints. We also investigated the effect of leptin on cartilage, using intraarticular injections of leptin in rats.. Leptin levels in SF samples obtained from OA patients undergoing either knee replacement surgery or knee arthroscopy were measured by enzyme-linked immunosorbent assay. In addition, histologic sections of articular cartilage and osteophytes obtained during surgery for total knee replacement were graded using the Mankin score, and were immunostained using antibodies to leptin, transforming growth factor beta (TGFbeta), and insulin-like growth factor 1 (IGF-1). For experimental studies, various doses of leptin (10, 30, 100, and 300 microg) were injected into the knee joints of rats. Tibial plateaus were collected and processed for proteoglycan synthesis by radiolabeled sulfate incorporation, and for expression of leptin, its receptor (Ob-Rb), and growth factors by reverse transcriptase-polymerase chain reaction and immunohistochemical analysis.. Leptin was observed in SF obtained from human OA-affected joints, and leptin concentrations correlated with the body mass index. Marked expression of the protein was observed in OA cartilage and in osteophytes, while in normal cartilage, few chondrocytes produced leptin. Furthermore, the pattern and level of leptin expression were related to the grade of cartilage destruction and paralleled those of growth factors (IGF-1 and TGFbeta1). Animal studies showed that leptin strongly stimulated anabolic functions of chondrocytes and induced the synthesis of IGF-1 and TGFbeta1 in cartilage at both the messenger RNA and the protein levels.. These findings suggest a new peripheral function of leptin as a key regulator of chondrocyte metabolism, and indicate that leptin may play an important role in the pathophysiology of OA.

Topics: Aged; Aged, 80 and over; Animals; Cartilage, Articular; Dose-Response Relationship, Drug; Female; Humans; Immunohistochemistry; Injections, Intra-Articular; Insulin-Like Growth Factor I; Knee Joint; Leptin; Male; Middle Aged; Osteoarthritis; Rats; Rats, Wistar; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Synovial Fluid; Tibia; Transforming Growth Factor beta; Transforming Growth Factor beta1

To study the mechanism of osteophyte formation in the ankle joints of adjuvant arthritic (AA) rats, the localization of peripheral nerves and immune cells in the synovia were investigated in both axotomized AA rats, whose sciatic nerves were resected before adjuvant injection, and sham-operated ones, using immunohistochemistry for low-affinity nerve growth factor receptor (p75NGFR), growth-associated protein (GAP)-43, calcitonin gene-related peptide (CGRP), helper T cell (W3/25), monocyte/macrophage (ED1), and transforming growth factor (TGF)-beta1 and its receptor, TGF-betaRII. In sham-operated AA rats, dense plexuses of CGRP-positive fibers were observed in the inflamed synovia close to the osteophytes. Most of the CGRP-positive fibers were also positive for p75NGFR and GAP-43. These fibers appeared to be newly sprouted sensory nerves. In axotomized AA rats, the synovia were supplied with no CGRP-positive fibers and the sizes of the osteophytes were smaller than those in sham-operated animals. The ratio of the number of both W3/25- and ED1-positive cells in the inflamed synovia of sham-operated rats peaked at weeks 2-3 after adjuvant injection. The peak, however, lasted until week 4 in axotomized ones. In both animal groups, the macrophages and the osteoblasts were stained for TGF-beta1. The osteoblasts covering the osteophytes were also stained for TGF-betaRII. The present findings suggest that the sensory nerves and the macrophages may be involved in osteophyte formation in the ankle joints of AA rats.

Topics: Animals; Ankle Joint; Arthritis, Experimental; Calcitonin Gene-Related Peptide; Female; GAP-43 Protein; Immune System; Immunohistochemistry; Macrophages; Neurons, Afferent; Osteoarthritis; Osteoblasts; Protein Serine-Threonine Kinases; Rats; Rats, Inbred Lew; Receptor, Nerve Growth Factor; Receptor, Transforming Growth Factor-beta Type II; Receptors, Nerve Growth Factor; Receptors, Transforming Growth Factor beta; Synovial Membrane; Transforming Growth Factor beta; Transforming Growth Factor beta1

The TGF-betas are multifunctional proteins whose activities are believed to be controlled by interaction with the latent TGF-beta binding proteins (LTBPs). In spite of substantial effort, the precise in vivo significance of this interaction remains unknown. To examine the role of the Ltbp-3, we made an Ltbp-3-null mutation in the mouse by gene targeting. Homozygous mutant animals develop cranio-facial malformations by day 10. At 2 mo, there is a pronounced rounding of the cranial vault, extension of the mandible beyond the maxilla, and kyphosis. Histological examination of the skulls from null animals revealed ossification of the synchondroses within 2 wk of birth, in contrast to the wild-type synchondroses, which never ossify. Between 6 and 9 mo of age, mutant animals also develop osteosclerosis and osteoarthritis. The pathological changes of the Ltbp-3-null mice are consistent with perturbed TGF-beta signaling in the skull and long bones. These observations give support to the notion that LTBP-3 is important for the control of TGF-beta action. Moreover, the results provide the first in vivo indication for a role of LTBP in modulating TGF-beta bioavailability.

Topics: Adaptor Proteins, Signal Transducing; Animals; Bone and Bones; Bone Remodeling; Carrier Proteins; Craniofacial Abnormalities; Gene Deletion; Gene Targeting; In Situ Hybridization; Latent TGF-beta Binding Proteins; Mice; Mice, Knockout; Osteoarthritis; Osteosclerosis; RNA, Messenger; Skull; Transforming Growth Factor beta

Aggrecanases are key matrix-degrading enzymes that act by cleaving aggrecan at the Glu(373)-Ala(374) site. While these fragments have been detected in osteoarthritis (OA) and rheumatoid arthritis (RA) cartilage and synovial fluid, no information is available on the regulation or expression of the two key aggrecanases (aggrecanase-1 and aggrecanase-2) in synovial tissue (ST) or fibroblast-like synoviocytes (FLS). The aggrecanase-1 gene was constitutively expressed by both RA and OA FLS. Real-time PCR demonstrated that TGF-beta significantly increased aggrecanase-1 gene expression in FLS. Aggrecanase-1 induction peaked after 24 h of TGF-beta stimulation. The expression of aggrecanase-1 mRNA was significantly greater in RA ST than in OA or nonarthritis ST. Aggrecanase-2 mRNA and protein were constitutively produced by nonarthritis, OA, and RA FLS but were not increased by IL-1, TNF-alpha, or TGF-beta. Furthermore, OA, RA, and nonarthritis ST contained similar amounts of immunoreactive aggrecanase-2. The major form of the aggrecanase-2 enzyme was 70 kDa in nonarthritis ST, whereas a processed 53-kDa form was abundant in RA ST. Therefore, aggrecanase-1 and -2 are differentially regulated in FLS. Both are constitutively expressed, but aggrecanase-1 is induced by cytokines, especially TGF-beta. In contrast, aggrecanase-2 protein may be regulated by a post-translational mechanism in OA and RA ST. Synovial and FLS production of aggrecanase can contribute to cartilage degradation in RA and OA.

Topics: ADAM Proteins; ADAMTS4 Protein; ADAMTS5 Protein; Amino Acid Sequence; Animals; Arthritis, Rheumatoid; Binding Sites, Antibody; Cattle; Cells, Cultured; Cytokines; Dose-Response Relationship, Immunologic; Enzyme Activation; Fibroblasts; Gene Expression Regulation; Humans; Immune Sera; Metalloendopeptidases; Molecular Sequence Data; Osteoarthritis; Procollagen N-Endopeptidase; RNA, Messenger; Synovial Membrane; Time Factors; Transforming Growth Factor beta

Transforming growth factor (TGF)-beta1 is expressed abundantly in the rheumatoid synovium. In this study, the inflammatory effect of TGF-beta1 in rheumatoid arthritis (RA) was investigated using cultured fibroblast-like synoviocytes (FLS) from RA and osteoarthritis (OA) patients, as well as non-arthritic individuals. mRNA expressions of IL-1beta, tumour necrosis factor (TNF)-alpha, IL-8, macrophage inflammatory protein (MIP)-1alpha and metalloproteinase (MMP)-1 were increased in RA and OA FLS by TGF-beta1 treatment, but not in non-arthritic FLS. Enhanced protein expression of IL-1beta, IL-8 and MMP-1 was also observed in RA FLS. Moreover, TGF-beta1 showed a synergistic effect in increasing protein expression of IL-1beta and matrix metalloproteinase (MMP)-1 with TNFalpha and IL-1beta, respectively. Biological activity of IL-1 determined by mouse thymocyte proliferation assay was also enhanced by 50% in response to TGF-beta1 in the culture supernatant of RA FLS. DNA binding activities of nuclear factor (NF)-kappaB and activator protein (AP)-1 were shown to increase by TGF-beta1 as well. These results suggest that TGF-beta1 contributes for the progression of inflammation and joint destruction in RA, and this effect is specific for the arthritic synovial fibroblasts.

Topics: Arthritis, Rheumatoid; Cells, Cultured; Chemokine CCL3; Chemokine CCL4; Cytokines; Fibroblasts; Humans; Interleukin-1; Interleukin-8; Macrophage Inflammatory Proteins; Matrix Metalloproteinase 1; NF-kappa B; Osteoarthritis; RNA, Messenger; Synovial Membrane; Transcription Factor AP-1; Transcriptional Activation; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

We investigated whether chondrocytes derived from osteoarthritic cartilage may lose their responsiveness to cartilage-derived morphogenetic protein-1, -2 (CDMP-1, -2) and osteogenic protein-1 (OP-1) compared with healthy cells, thus leading to an impaired maintenance of matrix integrity.. Chondrocytes were isolated from articular cartilage from patients with and without osteoarthritic lesions. Cells were grown as monolayer cultures for 7 days in a chemically defined serum-free basal medium (BM) in the presence of recombinant CDMP-1, -2, and OP-1. Glycosaminoglycan synthesis was measured by [35S]Sulfate incorporation into newly synthesized macromolecules. Cell proliferation was investigated by [3H]Thymidine incorporation. The endogenous gene expression of CDMPs/OP-1 and their respective type I and type II receptors was examined using RT-PCR. The presence of CDMP proteins in tissue and cultured cells was detected by Western immunoblots.. mRNAs coding for CDMPs and their respective receptors are endogenously expressed not only in healthy, but also in osteoarthritic cartilage. CDMP proteins are present in both normal and osteoarthritic articular cartilage and cultured chondrocytes. CDMP-1, CDMP-2 and OP-1 markedly increased glycosaminoglycan synthesis in both healthy (P< 0.01) and osteoarthritic (P< 0.05) human articular chondrocytes. A comparison of the glycosaminoglycan biosynthetic activity between healthy and osteoarthritic samples revealed no detectable difference, neither in stimulated nor in unstimulated cultures. [(3)H]Thymidine incorporation showed that CDMPs/OP-1 did not affect cell proliferation in vitro.. CDMPs and OP-1 exert their anabolic effects on both healthy and osteoarthritic chondrocytes indicating no loss in responsiveness to these growth factors in OA. The endogenous expression of CDMPs/OP-1 and their receptors suggest an important role in cartilage homeostasis.

Topics: Adult; Aged; Aged, 80 and over; Aggrecans; Blotting, Northern; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Cartilage, Articular; Cell Culture Techniques; Chondrocytes; Collagen Type II; Extracellular Matrix; Extracellular Matrix Proteins; Gene Expression Regulation; Growth Differentiation Factor 5; Growth Substances; Humans; Lectins, C-Type; Middle Aged; Osteoarthritis; Proteoglycans; RNA, Messenger; Transforming Growth Factor beta

To determine the capacity of human subchondral osteoarthritic osteoblasts (Ob) to produce interleukin (IL)-1beta, IL-6, transforming growth factor-beta (TGF-beta) and prostaglandin E(2) (PGE(2)), and determine if a relationship exists between IL-1beta, TGF-beta, PGE(2) and IL-6 production.. We measured the abundance of IL-1beta, IL-6, TGF-beta and PGE(2) using very sensitive ELISA in conditioned-media of human primary subchondral Ob from normal individuals and osteoarthritic patients. Selective inhibition of IL-6 or IL-6 receptor signaling was performed to determine its effect on PGE(2) production whereas the inhibiton of PGE(2) production was performed to determine its effect on IL-6 production. The expression of bone cell markers and urokinase plasminogen activator (uPA) activity was also determined.. Osteoarthritic Ob produced all these factors with greater variability than normal cells. Interestingly, the production of IL-6 and PGE(2) by osteoarthritic Ob separated patients into two subgroups, those whose Ob produced levels comparable to normal (low producers) and those whose Ob produced higher levels (high producers). In those cells classified as high osteoarthritic Ob, PGE(2) and IL-6 levels were increased two- to three-fold and five- to six-fold, respectively, compared with normal. In contrast, while using their IL-6 and PGE(2) production to separate osteoarthritic Ob into low and high producers, we found that IL-1beta levels were similar in normal and all osteoarthritic Ob. Using the same criteria, TGF-beta levels were increased in all osteoarthritic Ob compared with normal. Reducing PGE(2) synthesis by Indomethacin [a cyclo-oxygenase (COX) -1 and -2 inhibitor] reduced IL-6 levels in all osteoarthritic Ob, whereas Naproxen (a more selective COX-2 inhbitor) reduced PGE(2) and IL-6 levels only in the high osteoarthritic group. Conversely, PGE(2) addition to osteoarthritic Ob enhanced IL-6 production in both groups. Moreover, the addition of parathyroid hormone also stimulated IL-6 production to similar normal levels in both osteoarthritic groups. In contrast, using an antibody against IL-6 or IL-6 receptors did not reduce PGE(2) levels in either group. The evaluation of alkaline phosphatase activity, osteocalcin release, collagen type I and uPA activity in osteoarthritic Ob failed to show any differences between these cells regardless to which subgroup they were assigned.. These results indicate that IL-6 and PGE(2) production by subchondral Ob can discriminate two subgroups of osteoarthritic patients that cannot otherwise be separated by their expression of cell markers, and that endogenous PGE(2) levels influence IL-6 synthesis in osteoarthritic Ob.

Topics: Aged; Case-Control Studies; Cells, Cultured; Dinoprostone; Enzyme-Linked Immunosorbent Assay; Female; Humans; Interleukin-1; Interleukin-6; Male; Osteoarthritis; Osteoblasts; Phenotype; Transforming Growth Factor beta

We examined the production of substance P (SP) in synovial fibroblasts derived from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Immunoreactive SP was observed in non-stimulated RA fibroblasts. The expression of beta-preprotachykinin-A (beta-PPT-A) mRNA was confirmed by reverse transcription-polymerase chain reaction analysis. SP contents in culture medium were increased by treatment of RA fibroblasts with transforming growth factor-beta (TGFbeta) (10 ng/ml). Levels of SP release were elevated at 12 h after TGFbeta stimulation whereas the expression of beta-PPT-A mRNA was enhanced at 3 h. Furthermore, SP production in response to TGFbeta was dose-dependently enhanced by basic fibroblast growth factor (bFGF). OA fibroblasts also significantly released SP in the presence of TGFbeta (10 ng/ml) plus bFGF (50 ng/ml). These results suggest that SP produced by synovial fibroblasts may participate in joint diseases.

Topics: Arthritis, Rheumatoid; Cells, Cultured; Dose-Response Relationship, Drug; Fibroblast Growth Factor 2; Fibroblasts; Humans; Immunohistochemistry; Nerve Fibers; Osteoarthritis; Protein Precursors; RNA, Messenger; Substance P; Synovial Membrane; Tachykinins; Transforming Growth Factor beta

Recent data indicate that TGFbeta3, one member of the TGFbeta-isoforms, has an important role in bone remodeling. Up to date little is known about the expression and regulation of TGFbeta3 in man. We established a highly specific ELISA for quantitative measurement of TGFbeta3 in bone and blood samples and a RT-PCR in combination with HPLC for detection and quantification of TGFbeta3 mRNA in 89 human bone samples. Levels of TGFbeta3 protein ranged between 30 and 66 pg/mg bone (mean 36,6 +/-1,03 pg/mg) and between 30 and 1910 pg/ml in serum (mean 128.9+/-38.9 pg/ml). TGFbeta3 mRNA expression as well as protein levels in serum and in bone declined age dependently. No specific load- or site-specific distribution of TGFbeta3 mRNA expression or protein content was detected at different sites indicating an absence of mechanical regulation. Protein levels of TGFbeta3 in serum correlated with TGFbeta3 mRNA expression in bone (p= 0.0027; r=0.49). By contrast, TGFbeta3 protein levels stored in the bone matrix were not related to TGFbeta3 mRNA reflecting the long term process of TGFbeta3 deposition during bone remodeling. Notably TGFbeta3 serum levels were highly correlated with IGF-I and osteocalcin levels in serum. We conclude that TGFbeta3 in man circulates in significant amounts which appears to be representative for TGFbeta3 expression in bone tissue and may be in part derived from bone. The high correlation of TGFbeta3 with IGF-I suggests parallel systemic principles of regulation.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Aging; Bone and Bones; Female; Humans; Insulin-Like Growth Factor I; Male; Middle Aged; Osteoarthritis; Tissue Distribution; Transforming Growth Factor beta; Transforming Growth Factor beta3

Transforming growth factor beta is one of the most abundant growth factors stored in bone. It is known as a potent regulator of osteoblast proliferation and differentiation as well as of production extracellular matrix. We established a highly specific RT-PCR in combination with HPLC for detection and quantification of TGFbeta1 and TGFbeta2 mRNA expression in 89 human bone samples. Levels of TGFbeta1 protein ranged between 27 and 580 ng/g bone (mean 188 +/- 15 ng/g; n=75) and for TGFbeta2 between 7.2 and 35 ng/g bone (mean 14.3 +/- 2.1 ng/g; n=57). TGFbeta1 and TGFbeta2 protein concentrations and TGFbeta isoform mRNA expression in bone were not significantly different between the sexes. TGFbeta isoform mRNA expression as well as protein content in bone declined age dependently. TGFbeta1 and TGFbeta2 protein and mRNA expression were different in bone samples from different sites of the skeleton indicating in part the regulation by mechanical stimuli. In contrast to TGFbeta1, TGFbeta2 mRNA expression was significantly enhanced in osteoarthritic bone compared to unaffected bone. These data are in concordance to previous results concerning the expression of TGFbeta3 in bone. In conclusion, the data suggest distinct patterns' of expression of the TGFbeta isoforms under physiological and pathological conditions in bone.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Aging; Bone and Bones; Chromatography, High Pressure Liquid; Female; Fractures, Bone; Gene Expression; Humans; Male; Middle Aged; Osteoarthritis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta2

To examine the signaling pathways leading to transforming growth factor-beta (TGF-beta) induced collagenase-3 production in human osteoarthritic (OA) chondrocytes, as well as the transcription factors and their binding sites involved in the transcriptional control of collagenase-3 gene.. Identification of the TGF-beta signaling pathway was by Western immunoblotting using specific antibodies for the phosphorylated forms of p44/42 and p38 MAPK, SAPK/JNK, and the Smad2 protein. Electromobility shift assays (EMSA) were carried out for activator protein- (AP-1), polyomavirus enhancer A (PEA-3), activin-response-element-like, Smad-binding-element-like, and TGF-beta inhibitory element oligonucleotides. Supershift assays using antibodies to the Jun, Fos, and Smad families of proteins were used for identification of transcription factors. Chondrocyte transfections were also performed using the -133CAT collagenase-3 promoter plasmid (containing PEA-3, AP-1, and TATA sites) and mutated AP-1 and PEA-3 sites.. The primary target of TGF-beta induced collagenase-3 in OA chondrocytes was the Smad2 protein, with significant phosphorylation within 5 min. Contrasting with the Smad2, the untreated OA chondrocytes already had detectable levels of the phosphorylated forms of p38 and p44/42 MAPK. Of the oligonucleotides tested, EMSA revealed that TGF-beta treated OA chondrocyte proteins bound only to the AP-1 and PEA-3. Supershifts with the AP-1 oligonucleotide showed the presence of the Jun (c-Jun, JunB, JunD) and Fos (c-Fos, FosB, Fra-1, Fra-2) proteins in the untreated and TGF-beta treated OA chondrocytes, whereas only Smad proteins (Smad2, 3, 4) were present in the AP-1 binding proteins from the TGF-beta treated chondrocytes. The AP-1 mutation decreased both basal (95%) and TGF-beta induced (99%) collagenase-3 production, whereas the PEA-3 mutation decreased the basal (15%) but more significantly (50%) the TGF-beta induced transcription.. Smad proteins are the main cytoplasmic signaling pathways in TGF-beta stimulated collagenase-3 in OA chondrocytes. The AP-1 site appears critical for upregulation of collagenase-3 production, but TGF-beta stimulation requires both AP-1 and PEA-3 sites for optimal response.

Topics: Aged; Cartilage, Articular; Cells, Cultured; Chondrocytes; Collagenases; DNA-Binding Proteins; Enzyme Activation; Humans; Matrix Metalloproteinase 13; Middle Aged; Mitogen-Activated Protein Kinases; Mutagenesis; Osteoarthritis; Phosphorylation; Protein Binding; Signal Transduction; Smad2 Protein; Stimulation, Chemical; Trans-Activators; Transcription Factor AP-1; Transcription Factors; Transforming Growth Factor beta

Osteoarthritis (OA) is a degenerative joint disease characterised by the breakdown of the extracellular matrix of chondrocytes in the affected joints. Cytokines and growth factors which are known to play a role in the synthesis and degradation of cartilage matrix have been shown to be upregulated in osteoarthritic cartilage. This upregulation resulted in two different phenotypes, overexpressing either TNF-alpha and IL-6 or IL-1beta, TGFbeta1, IL-4 and IL-10. To investigate the hierarchy among growth factors and cytokines involved in cartilage metabolism, we analysed osteoarthritic cartilage explants for their responses to human recombinant (rh) cytokines and growth factors. The cytokine expression patterns of the explants before and after in vitro culture were compared by immunohistological staining of cartilage sections. We found a coordinate expression of TNF-alpha and IL-6 on the one hand, and of IL-1beta, TGFbeta1, IL-4 and IL-10 on the other. Although TNF-alpha and IL-6 stimulated each other's expression, they downregulated TGF beta1, IL-4 and IL-10 or IL-1beta, TGF beta1 and IFNdelta, respectively. IL upregulated the expression of TGF beta1, IL-4 and IL-10, and jointly these four cytokines and growth factors downregulated IL-6. Both of the expression patterns described for OA cartilage can be explained by these regulatory mechanisms. Interestingly, no cytokine efficiently downregulated TNF-alpha, and even though IL-1beta is upregulated in one of the OA phenotypes, none of the growth factors and cytokines tested--except for IL-1beta itself--seemed capable of mediating this upregulation. This unresponsiveness to cytokine stimulation might hint at a genetic cause for the elevated expression in the respective phenotypes.

Topics: Aged; Aged, 80 and over; Biopsy, Needle; Cartilage, Articular; Culture Techniques; Cytokines; Down-Regulation; Female; Growth Substances; Humans; Immunohistochemistry; Interleukin-10; Interleukin-4; Interleukin-6; Male; Middle Aged; Osteoarthritis; Prospective Studies; Sensitivity and Specificity; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Up-Regulation

To examine the impact of prolonged TGF-beta exposure on cartilage and ligamentous joint structures in vivo, to investigate involvement of TGF-beta in osteoarthritis pathology.. TGF-beta was injected into murine knee joints once or repeatedly, whereafter articular cartilage proteoglycan (PG) synthesis and content, and histological changes in knee joints were studied over a 2-month period.. A single injection of TGF-beta stimulated patellar cartilage PG synthesis for 3 weeks and PG content for 2 weeks. Triple TGF-beta injections prolonged the increase in PG content to 3 weeks. Patellar cartilage showed no histological abnormalities at 1 and 2 months after the last injection. In contrast, 2 months after triple TGF-beta injections the superficial layer of tibial cartilage still had an increased proteoglycan content, while severe PG depletion was found in deeper layers of the posterior part of the lateral tibia in particular. Eventually, lesions occurred at the level of the tide-mark, exactly the site where cartilage is torn off in experimental and spontaneous osteoarthritis in mice. Additionally, multiple TGF-beta injections induced formation of chondroid structures along the margins of articular cartilage. These chondroid structures were transformed into osteophytes via endochondral ossification. Formation of chondroid tissue was also observed in collateral ligaments.. Multiple intra-articular injections of TGF-beta induce changes in articular cartilage and surrounding tissues that have strong resemblance to features of experimental and spontaneous osteoarthritis in mice, suggesting a role for TGF-beta in the OA process.

Topics: Animals; Cartilage, Articular; Injections, Intra-Articular; Knee Joint; Male; Mice; Mice, Inbred C57BL; Osteoarthritis; Proteoglycans; Transforming Growth Factor beta

This study demonstrates for the first time that human articular chondrocytes express osteogenic protein-1 (OP-1). OP-1 was originally purified from bone matrix and was shown to induce cartilage and bone formation. Both OP-1 protein and message were present in human normal and osteoarthritic (OA) cartilages. OP-1 mRNA was upregulated in OA cartilage compared with normal adult tissues. However, the level of mature OP-1 protein in the same OA tissues was downregulated, whereas the pro-OP-1 remained high. Moreover, these two forms of OP-1 were localized in an inverted manner. Mature OP-1 was primarily detected in the superficial layer, whereas the pro-form was mostly in the deep layer of cartilage. The presence of pro- and mature OP-1 in extracts of normal and OA cartilages was confirmed by Western blotting. These findings imply that articular chondrocytes continue to express and synthesize OP-1 throughout adulthood. The observed patterns of the distribution of pro- and mature OP-1 also suggest differences in the processing of this molecule by normal and OA chondrocytes and by the cells in the superficial and deep layers. Distinct distribution of OP-1 and its potential activation in deep zones and regions of cloning in OA cartilages may provide clues to the potential involvement of endogenous OP-1 in repair mechanisms. (J Histochem Cytochem 48:239-250, 2000)

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Blotting, Western; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Cartilage, Articular; Child; Child, Preschool; Chondrocytes; Female; Humans; Immunohistochemistry; In Situ Hybridization; Infant; Infant, Newborn; Male; Middle Aged; Osteoarthritis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta

To correlate the increased collagenase production previously seen in chondrocytes isolated from osteoarthritic (OA) lesions and the expression of cytokines and cytokine receptors.. Chondrocytes were isolated from OA cartilage and characterized for synthesis of collagenases, cytokines, and cytokine receptors by Northern and Western blot analyses, RNA protection assay, and flow cytometry.. Chondrocytes located in cartilage proximal to the macroscopic OA lesions bound more tumor necrosis factor alpha (TNFalpha) and interleukin-1beta (IL-1beta) compared with chondrocytes isolated from morphologically normal cartilage from the same joint. In response to TNFalpha stimulation, messenger RNA (mRNA) levels for the IL-1 receptor I (IL-1RI), IL-1RII, TNF receptor II (TNFR II), and IL-6 receptor as well as the level of proinflammatory cytokines, such as IL-1alpha, IL-1beta, lymphotoxin beta, TNFalpha, and IL-6, also increased. In contrast, treatment with transforming growth factor beta1 (TGFbeta1) resulted in down-regulation of matrix metalloproteinase 1 (MMP-1) and MMP-13 concomitant with a reduction in the levels of mRNA for IL-1RI, IL-1RII, TNFRI, and TNFRII and proinflammatory cytokine levels. In contrast, the levels of mRNA for TGFbeta receptor I, TGFbeta1, and TGFbeta3 were up-regulated.. These data show that TGFbeta1 has antagonistic effects upon OA chondrocytes, in contrast to the effects seen with TNFalpha. The cyclical course of OA, where a period of active disease is followed by a period of remission, can be explained by a sequential pattern of cytokine stimulation followed by a feedback inhibition of autocrine cytokine production and cytokine receptor expression, thus affecting collagenase synthesis.

Topics: Aged; Autocrine Communication; Chondrocytes; Collagenases; Flow Cytometry; Gene Expression Regulation, Enzymologic; Humans; Interleukin 1 Receptor Antagonist Protein; Interleukin-1; Interleukin-10; Interleukin-6; Matrix Metalloproteinase 1; Matrix Metalloproteinase 13; Matrix Metalloproteinases; Middle Aged; Osteoarthritis; Phenotype; Receptors, Interleukin-1; Receptors, Interleukin-1 Type II; Receptors, Interleukin-6; Receptors, Tumor Necrosis Factor; RNA, Messenger; Sialoglycoproteins; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

To investigate the relationships between serum and urinary molecular markers (MM) used to monitor osteoarthritis.. Forty osteoarthritis patients had blood and urine collected at baseline and 1, 3, 6 and 12 months later. Specimens from 20 controls were obtained twice at a one month interval. The concentration of 14 different markers was determined at each time point and the data were analyzed by statistical methodology.. The markers could be divided by the method of principal components analysis into five clusters of related markers: inflammation markers (C-reactive protein, tumor necrosis receptor type I and tumor necrosis receptor type II, interleukin 6, eosinophilic cationic protein), bone markers (bone sialoprotein, hydroxylysyl pyridinoline, lysyl pyridinoline), putative markers of cartilage anabolism (carboxypropeptide of type II procollagen, hyaluronan, epitope 846) and catabolism (keratan sulfate, cartilage oligomeric matrix protein), and transforming growth factor beta. Three markers (tumor necrosis factor receptor II, cartilage oligomeric matrix protein and epitope 846) from independent clusters discriminated osteoarthritis patients from controls. Inflammation was not a confounding factor in measurement, but a recognizable distinguishing factor in osteoarthritis.. The markers separated into rational groups on the basis of their covariance, a finding with independent biochemical support. The covariance of markers from the same cluster suggests the use of a representative marker from the cluster to reflect changes in osteoarthritis. If multiple markers are being measured within a single cluster, then the use of a weighted cluster 'factor' may be preferable to the separate use of individual markers.

Topics: Amino Acids; Biomarkers; Blood Proteins; C-Reactive Protein; Carboxypeptidases; Case-Control Studies; Epitopes; Extracellular Matrix Proteins; Female; Humans; Hyaluronic Acid; Interleukin-6; Keratan Sulfate; Male; Middle Aged; Osteoarthritis; Osteoarthritis, Hip; Osteoarthritis, Knee; Procollagen; Receptors, Tumor Necrosis Factor; Sialoglycoproteins; Transforming Growth Factor beta

Osteoarthritic lesions appear in the articular cartilage of the temporomandibular joint of mice aged 7 months and older. Reduced rate of proteoglycan (PG) synthesis leading to destruction of the articular cartilage was observed in this joint. The purpose of the present study was to test the ability of transforming growth factor-beta1 (TGF-beta1), insulin-like growth factor-1 (IGF-1) and growth hormone (GH) to induce PG synthesis in joint cartilage of aged animals and to compare it with the effect of interleukin-1alpha (IL-1alpha). Mandibular condyle explants from 18-month-old mice were cultured up to 72 h in serum-free medium, supplemented with IL-1alpha (TGF-beta1 (0.1-5.0 ng/ml), TGF-beta1 (1.0 ng/ml) + IGF-1 (2 ng/ml) or GH (10 ng/ml). The incorporation of (35)S-SO(4) into sulfated PG was tested. Cartilage samples were processed for histomorphometry using sections stained with 0.1% toluidine blue (TB), pH 1.8. Results indicated that in cultures supplemented (48 h) with either TGF-beta, TGF-beta + IGF-1 or with GH, an increased height and area of TB-positive staining as well as increased incorporation of (35)S-SO(4) into sulfated PG were observed. In contrast, the cytokine IL-1alpha exerted an inhibitory effect on TB staining and on (35)S-SO(4) incorporation. The present study demonstrated that in vitro supplementation of IL-1alpha to mandibular condyle cartilage reduced the height and area of TB staining and incorporation of (35)S-SO(4), whereas TGF-beta1, TGF-beta1 + IGF-1 or GH increased the height and area of TB staining and increased incorporation of (35)S-SO(4). The two parameters used to identify increased PG synthesis were shown to reveal similar results and were useful for studying the dynamic events taking place in cartilage destruction and repair in osteoarthritis.

Topics: Age Factors; Animals; Cartilage, Articular; Coloring Agents; Female; Growth Hormone; Insulin-Like Growth Factor I; Interleukin-1; Mandibular Condyle; Mice; Mice, Inbred ICR; Organ Culture Techniques; Osteoarthritis; Proteoglycans; Staining and Labeling; Temporomandibular Joint; Tolonium Chloride; Transforming Growth Factor beta

Osteoporosis and osteoarthritis each exhibit a strong genetic component. Although polymorphisms of a variety of genes have been associated with bone mineral density and genetic susceptibility to osteoporosis or to osteoarthritis, the genes responsible for these conditions have not been definitively identified. We have shown that a T(869)-->C polymorphism of the transforming growth factor-beta1 (TGF-beta1) gene, which results in a Leu-->Pro substitution at amino acid 10, is associated with bone mineral density in Japanese adolescents and postmenopausal women, with genetic susceptibility to osteoporosis or spinal osteoarthritis, and with the outcome of treatment for osteoporosis with active vitamin D. I here review our recent studies, which have provided insight into the function of TGF-beta1 as well as into the role of genetic factors in the development of osteoporosis and osteoarthritis.

Topics: Animals; Bone Density; Disease Susceptibility; Genetic Predisposition to Disease; Genotype; Humans; Leucine; Osteoarthritis; Osteoporosis; Polymorphism, Genetic; Proline; Radius; Transforming Growth Factor beta; Transforming Growth Factor beta1; Treatment Outcome

Morphologic changes observed in osteoarthritis include cartilage erosion and a variable degree of synovial inflammation. Proinflammatory cytokines such as interleukin-1 beta, locally produced by the inflamed synovium, also likely contribute to these alterations. Despite an extensive armamentarium and numerous surgical options, osteoarthritis remains incurable, and an improved approach in the treatment of this disease is imperative. Drug delivery is a major weakness of existing antiarthritic therapies. Local delivery of antiinflammatory cytokines or the in vivo induction of their expression using gene transfer may provide a novel approach for the treatment of osteoarthritis. Evidence of the efficacy of gene therapy in osteoarthritis remains very scarce. To the authors' knowledge, there is no clinical research protocol en route for the treatment of osteoarthritis using gene therapy. The authors present the only two studies that have proved successful in treating animal models of osteoarthritis using gene therapy, and propose an overview of several strategies for the development of gene therapy in osteoarthritis treatment in the future.

Topics: Animals; Forecasting; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Humans; Interleukin-1; Osteoarthritis; Transforming Growth Factor beta

To investigate the ability of doxycycline, transforming growth factor beta1 (TGFbeta1), and phorbol myristate acetate (PMA) to modulate collagenase synthesis in osteoarthritic (OA) chondrocytes.. Levels of fibroblast collagenase (matrix metalloproteinase 1 [MMP-1]), neutrophil collagenase (MMP-8), and collagenase 3 (MMP-13) proteins and messenger RNA (mRNA) were measured in chondrocytes isolated from involved and uninvolved areas of OA cartilage and from normal human chondrocytes, after treatment with doxycycline, TGFbeta1, and PMA.. Chondrocytes isolated from cartilage immediately adjacent to the OA lesion had, on average, 1.8-3.9-fold higher basal levels of MMP mRNA. These cells down-regulated collagenase proteins and mRNA upon incubation with TGFbeta1. In contrast, chondrocytes from areas located more distant from the macroscopic lesion increased MMP-13 mRNA, while MMP-1 and MMP-8 decreased after stimulation with TGFbeta1. Discoordinate regulation was observed after stimulation with PMA, with an increase in MMP-1 and MMP-8 but a decrease in MMP-13. Incubation of OA chondrocytes with doxycycline (1-10 microg/ml), at pharmacologically achievable levels, decreased levels of mRNA of all 3 collagenases, but not G3PDH. In addition, doxycycline inhibited the increase in mRNA for these enzymes in normal chondrocytes stimulated with tumor necrosis factor alpha.. These findings suggest that regulation of MMP-1, MMP-8, and MMP-13 in OA chondrocytes, although mediated by differing pathways, can be decreased by treatment with doxycycline at low concentrations. Our data provide a rationale for the use of doxycycline in the treatment of OA.

Topics: Aged; Anti-Bacterial Agents; Blotting, Northern; Blotting, Western; Carcinogens; Cells, Cultured; Chondrocytes; Collagenases; Doxycycline; Gene Expression Regulation, Enzymologic; Humans; Matrix Metalloproteinase 1; Matrix Metalloproteinase 13; Matrix Metalloproteinase 8; Middle Aged; Osteoarthritis; RNA, Messenger; Synovial Membrane; Tetradecanoylphorbol Acetate; Transforming Growth Factor beta

Diacetylrhein or diacerein has shown efficacy in the treatment of both major forms of osteoarthritis (OA), coxarthrosis as well as gonarthrosis, improving clinical symptoms of the disease (pain reduction and algo-functional index). Both in-vitro and animal models studies suggest that diacerein may have also disease-modifying effects. The drug exerts inhibitory effects on interleukin-1-induced expression of cartilage degrading enzymes. However, its mechanism of action is not completely understood. In view of the role that could play the transforming growth factor (TGF)-beta system in the repair potentialities of OA cartilage, we studied the effect of diacerein on the expression of TGF-beta isoforms 1, 2 and 3 and that of their receptor types I and II in cultured bovine chondrocytes.. Cultured bovine articular chondrocytes were treated with 10(-5) m diacerein, 10 ng/ml IL-1beta or the combination diacerein+interleukin (IL)-1, and the expression of both TGF-beta isoforms 1, 2 and 3 and that of their receptors TbetaR-I and TbetaR-II was determined by Northern-blot and reverse transcriptase-polymerase chain reaction (RT-PCR). Cell transfections of cDNA constructs containing sequences of the 5'-upstream region of TGF-beta1 promoter were also performed to determine their transcriptional activity in diacerein-treated cultures.. The data indicated that diacerein enhances the expression of TGF-beta1 and TGF-beta2. This effect was also found in the presence of IL-1, albeit with smaller intensity. In contrast, the levels of TGF-beta3 and receptors I and II remained unaffected or slighty modified by the compound. Treatment of cells transiently transfected with TGF-beta1 promoter constructs suggested that the stimulating effect on TGF-beta1 expression is mediated by the region -1038 to -1132 base pars.. The results suggest that diacerein effects on matrix synthesis and turn-over previously reported in cultured articular chondrocytes might be explained in part by the ability of the drug to enhance TGF-beta1 and TGF-beta2 expression in these cells. This mechanism of action may account for the potential disease-modifying properties of diacerein and might give clues as to how future anti-osteoarthritic drugs should be designed.

Topics: Animals; Anthraquinones; Anti-Inflammatory Agents, Non-Steroidal; Cartilage; Cattle; Chondrocytes; Drug Evaluation, Preclinical; Luciferases; Osteoarthritis; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; Transfection; Transforming Growth Factor beta

Topics: Cartilage; Chondrocytes; Disease Progression; Growth Substances; Humans; Osteoarthritis; Transforming Growth Factor beta

Topics: Bone and Bones; Bone Remodeling; Chondrocytes; Humans; Insulin-Like Growth Factor I; Osteoarthritis; Sclerosis; Transforming Growth Factor beta

Topics: Animals; Bone and Bones; Bone Development; Cartilage, Articular; Disease Models, Animal; Mice; Osteoarthritis; Osteocytes; Transforming Growth Factor beta

We investigated the response of human osteoarthritic (OA) chondrocytes, in terms of collagenase 3 production, to growth factors and cytokines involved in the anabolism and catabolism of articular cartilage, and explored the major signaling pathways leading to its up-regulation.. Human OA chondrocytes were treated with the following factors: the proinflammatory cytokine interleukin-1beta (IL-1beta), the growth factors basic fibroblast growth factor (bFGF), platelet-derived growth factor BB (PDGF-BB), parathyroid hormone (PTH), insulin-like growth factor 1 (IGF-1), transforming growth factor gamma1 (TGFbeta1), and TGFbeta2, the protein kinase (PK) activator antagonists for PKC, PKA, and PKG pathways, and phospholipase A2 and tyrosine kinases, as well as the antiinflammatory cytokines IL-4, IL-10, and IL-13. Collagenase 3 expression and synthesis were determined. Comparison was made with collagenase 1.. The human OA chondrocyte population could be divided into 2 categories: the L chondrocytes, showing low collagenase 3 basal synthesis levels and high sensitivity to IL-1beta stimulation; and the H chondrocytes, high collagenase 3 basal synthesis levels and low IL-1beta inducibility. In L chondrocytes, all growth factors stimulated collagenase 3 production. In H chondrocytes, PTH, IGF-1, and TGFbeta had little or no impact; bFGF slightly stimulated it and PDGF-BB showed the same pattern as in the L chondrocytes. The effects of all growth factors, except TGFbeta, on collagenase 1 synthesis followed those of collagenase 3, albeit to a higher degree. Interestingly and unlike collagenase 3, the effects of TGFbeta on collagenase 1 could not be related to the state of the cells, but rather, depended on the isoform. Indeed, TGFbeta2 did not induce collagenase 1 synthesis, whereas TGFbeta1 stimulated it. Among the PK activators tested, phorbol myristate acetate was the strongest inducer, suggesting a major involvement of the PKC pathway. IL-13 inhibited collagenase 3 production, IL-4 had little effect, and IL-10 had none.. This study shows that collagenase 3 production in human OA chondrocytes depends on the physiologic state of the cell. TGFbeta might be responsible for the change in cells from the L to the H state. Importantly, our in vitro data implicate TGFbeta2 as a possible in vivo agent capable of specifically triggering collagenase 3 production over that of collagenase 1 in OA cartilage.

Topics: Aged; Cartilage, Articular; Cells, Cultured; Chondrocytes; Collagenases; Cytokines; Down-Regulation; Female; Growth Substances; Humans; Male; Matrix Metalloproteinase 1; Matrix Metalloproteinase 13; Osteoarthritis; Protein Kinase C; Signal Transduction; Tetradecanoylphorbol Acetate; Transforming Growth Factor beta; Up-Regulation

We investigated gene expression of antiinflammatory mediators in the interfacial membranes retrieved at hip revision arthroplasty using reverse transcription-polymerase chain reaction (RT-PCR). Levels of RT-PCR products were compared with those of synovial tissue from patients with osteoarthrosis or rheumatoid arthritis. Antiinflammatory mediators such as type II interleukin (IL)-1 receptor, IL-4, IL-10, IL-1 receptor antagonist, and transforming growth factor-beta 1 (TGF-beta 1) were expressed in the interfacial membrane. In interfacial tissue, the level of IL-10 was lower, but that of the IL-1 receptor antagonist higher than in diseased synovial tissue.

Topics: Adult; Aged; Aged, 80 and over; Arthritis, Rheumatoid; Case-Control Studies; Female; Gene Expression; Hip Prosthesis; Humans; Inflammation; Inflammation Mediators; Interleukin 1 Receptor Antagonist Protein; Interleukin-10; Interleukin-4; Male; Middle Aged; Osseointegration; Osteoarthritis; Prosthesis Failure; Receptors, Interleukin-1; Reoperation; Reverse Transcriptase Polymerase Chain Reaction; Sialoglycoproteins; Synovial Membrane; Transforming Growth Factor beta

This study was undertaken to examine the presence of interleukin-1 receptor antagonist (IL-1ra), IL-10, and transforming growth factor-beta1 (TGF-beta1) in the synovial fluid (SF) lavage specimens of patients with temporomandibular disorders (TMDs).. Synovial fluid lavage specimens were obtained from 14 temporomandibular joints (TMJs) of 12 patients with TMJ internal derangement (ID) and 17 TMJs of 15 patients with TMJ osteoarthritis (OA). Seven synovial fluid lavage samples of TMJs of four asymptomatic donors served as normal controls. The concentrations of IL-1ra, IL-10, and TGF-beta1 were detected with sensitive and specific sandwich enzyme-linked immunosorbent assay (sandwich-ELISA).. IL-1ra, IL-10, and TGF-beta1 in all the normal controls were undetectable. IL-1ra concentrations were 175.78 +/- 52.43 pg/mL in the patients with TMJ ID and 187.85 +/- 59.51 pg/mL in those with TMJ OA. IL-10 was undetectable in all the TMJ ID and OA samples. The concentration of TGF-beta1 in TMJ ID patients (47.93 +/- 88.25 pg/mL) was significantly less than in patients with TMJ OA (143.61 +/- 108.00 pg/mL) (P < .01).. The results suggest that deficiencies of IL-1ra, IL-10, and TGF-beta1 probably play an important role in the cause and pathogenesis of TMJ ID and OA.

Topics: Adolescent; Adult; Chronic Disease; Enzyme-Linked Immunosorbent Assay; Female; Humans; Interleukin 1 Receptor Antagonist Protein; Interleukin-10; Male; Middle Aged; Osteoarthritis; Receptors, Interleukin-1; Sensitivity and Specificity; Sialoglycoproteins; Synovial Fluid; Temporomandibular Joint; Temporomandibular Joint Disorders; Therapeutic Irrigation; Transforming Growth Factor beta

Topics: Humans; Interleukin-1; Osteoarthritis; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

The purpose of this study was to analyze the expression of bone morphogenetic protein-2 (BMP-2) in patients with internal derangement of the temporomandibular joint.. Twenty-one human temporomandibular joint samples (5 extirpated disks and 16 biopsy specimens of synovitis area from patients with internal derangement of the TMJ) and 2 control temporomandibular joint specimens (2 normal disks obtained by autopsy) were analyzed with specific antibodies through use of an immunohistochemical technique.. BMP-2 was predominantly localized in chondrocytes around the damaged areas of the articular disks. BMP-2 expression was also found in synovial cells and endothelial cells of blood vessels. Control specimens demonstrated BMP-2 staining in synovial lining cells and endothelial cells of blood vessels. However, the chondrocytes in the normal cartilage layers of the control specimens showed no staining.. These findings suggest that BMP-2 may be involved in the pathogenesis of osteoarthritic changes or the repair process of temporomandibular joint internal derangement.

Topics: Adult; Biopsy; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Humans; Immunohistochemistry; Osteoarthritis; Synovial Membrane; Temporomandibular Joint; Temporomandibular Joint Disc; Temporomandibular Joint Disorders; Transforming Growth Factor beta

Biochemical investigations into the pathogenesis of osteoarthritis have, for the last two decades, concentrated on the mechanisms involved in the destruction of the articular cartilage. Although bone changes are known to occur, the biochemistry of the collagenous matrix within osteoarthritic bone has received scant attention. We report that bone collagen metabolism is increased within osteoarthritic femoral heads, with the greatest changes occurring within the subchondral zone. Collagen synthesis and its potential to mineralize were determined by the carboxy-terminal propeptide content and alkaline phosphatase activity, respectively. These data supported elevated new matrix formation. Our finding of a three- to fourfold increase in TGF-beta in osteoarthritic bone indicates that this might represent a stimulus for the increased collagen synthesis observed. Of additional significance is the hypomineralization of deposited collagen in the subchondral zone of osteoarthritic femoral heads, supporting a greater proportion of osteoid in the diseased tissue. The cross-linking of collagen was similar to that observed for controls. In addition, the degradative potential of osteoarthritic bone was considerably higher as demonstrated by increased matrix metalloproteinase 2 activity, and again the greater activity was associated with the subchondral bone tissue. The polarization exhibited in the metabolism of bone collagen from osteoarthritic hips might exacerbate the processes involved in joint deterioration by altering joint morphology. This in turn may alter the distribution of mechanical forces to the various tissues, to which bone is a sensitive responder. Bone collagen metabolism is clearly an important factor in the pathogenesis of osteoarthritis and certainly warrants further biochemical study.

Topics: Aged; Aged, 80 and over; Alkaline Phosphatase; Amino Acids; Bone and Bones; Bone Density; Case-Control Studies; Collagen; Cross-Linking Reagents; Female; Femur Head; Gelatinases; Humans; Matrix Metalloproteinase 2; Metalloendopeptidases; Osteoarthritis; Solubility; Transforming Growth Factor beta

To investigate factors regulating fibronectin fibrillar matrix formation by synovial fibroblasts.. Basal and cytokine stimulated extracellular matrix (ECM) fibronectin produced by synovial fibroblasts was identified by immunofluorescence and Western blot. Alternative mRNA splicing of fibronectin was studied by reverse transcription polymerase chain reaction. The integrin receptor responsible for supporting fibronectin fibrillar matrix was identified by blocking antibodies and receptor levels studied by Western blot.. Transforming growth factor-beta (TGF-beta) or platelet derived growth factor (PDGF), but not interleukin 1 or exogenous fibronectin, induced ECM fibronectin. ECM fibronectin was blocked by the addition of antibody to the alpha5beta1 integrin. Cytokines did not significantly change alternative mRNA splicing of fibronectin or levels of alpha5beta1 integrin expression.. Synovial cell production of a fibrillar fibronectin matrix is induced by growth factors, including TGF-beta and PDGF. This induction is mediated by the alpha5beta1 integrin. Since fibrillar fibronectin formation was not strongly dependent on increased fibronectin or alpha5beta1 integrin levels, this effect may be mediated by growth factor induced changes in receptor affinity.

Topics: Alternative Splicing; Anti-Allergic Agents; Antibodies, Monoclonal; Arthritis, Rheumatoid; Blotting, Northern; Blotting, Western; Extracellular Matrix; Fibroblasts; Fibronectins; Fluorescent Antibody Technique, Direct; Humans; Integrin alpha4beta1; Integrins; Interleukin-1; Osteoarthritis; Platelet-Derived Growth Factor; Receptors, Fibronectin; Receptors, Lymphocyte Homing; RNA, Messenger; Synovial Membrane; Transforming Growth Factor beta

Activin A is a cytokine whose multiple functions have yet to be fully determined. In this study, the role of proinflammatory cytokines in regulatory control of activin A production was shown in synoviocytes and chondrocytes. Additional facets of functional inflammation-related activities of activin A were also determined. Results showed that activin A concentrations in the synovial fluid of patients with rheumatoid arthritis and gout were elevated relative to those in patients with osteoarthritis. Further studies showed that production of activin A by synoviocytes and chondrocytes in culture was stimulated by cytokines such as IL-1, transforming growth factor-beta (TGF-beta), interferon-gamma (IFN-gamma), and IL-8, consistent with previous studies in regard to the control of activin A production in marrow stromal cells and monocytes by cytokines, glucocorticoids and retinoic acid. In addition, the relationship of activin A to IL-6-induced biological activities was investigated. Three major IL-6 activities involved in inflammatory responses were found to be suppressed by activin A. In a dose-dependent manner, activin A efficiently suppressed IL-6-induced proliferation of 7TD1 B lymphoid cells, phagocytic activity of monocytic M1 cells, and fibrinogen production in HepG2. Therefore, it is likely that activin A serves as a suppressor for IL-6, dampening inflammatory responses, and has the potential to perform some previously unrecognized roles in inflammation.

Topics: Activins; Arthritis, Rheumatoid; Chondrocytes; Humans; Inhibins; Interferon-gamma; Interleukin-1; Interleukin-6; Osteoarthritis; Synovial Fluid; Synovial Membrane; Transforming Growth Factor beta

Topics: Animals; Cartilage, Articular; Hindlimb; Joints; Male; Osteoarthritis; Polymerase Chain Reaction; Protein Serine-Threonine Kinases; Rabbits; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; RNA, Messenger; Time Factors; Transforming Growth Factor beta

Rheumatoid arthritis and periodontitis are chronic inflammatory diseases associated with tissue destruction that is mediated in part by elevated levels of cytokines (e.g., interleukin-1 and tumor necrosis factor). Differential screening of a human synovial fibroblast cDNA library for interleukin-1 induced genes revealed a clone identical to the gene encoding human bone morphogenetic protein-2. Northern blot analysis of human synovial fibroblast mRNA confirmed up-regulation of bone morphogenetic protein-2 in the presence of interleukin-1. Utilizing a specific antibody, levels of bone morphogenetic protein-2 protein in conditioned medium from synovial fibroblasts were also up-regulated in the presence of interleukin-1. This is the first report of the production of bone morphogenetic protein-2 by synovial fibroblasts, and the first report of its up-regulation in response to interleukin-1. However, interleukin-1 did not induce bone morphogenetic protein-2 mRNA in human gingival fibroblasts.

Topics: Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cells, Cultured; Culture Media, Conditioned; Fibroblasts; Gene Expression Regulation; Gene Library; Gingiva; Humans; Interleukin-1; Osteoarthritis; Protein Biosynthesis; RNA, Messenger; Synovial Membrane; Transcription, Genetic; Transforming Growth Factor beta

To clarify in vivo applicability of adenovirus mediated gene delivery to examine a gene therapy for human joint diseases.. We directly injected vectors harbouring beta-galactosidase gene and transforming growth factor (TGF)-beta1 gene into the joints of Hartley guinea pigs. Expressions of delivered LacZ were examined by 5-bromo-4-chloro-3-indolyl-beta-D-galactoside staining and reverse transcription-polymerase chain reaction. The levels of TGF-beta1 that were delivered to the joint and then transferred to the joint fluid were assessed by ELISA.. LacZ expression was observed in almost all synovial tissue samples and in chondrocytes on the surface of degenerated cartilage. In the other organs, expression of delivered genes was not observed. For 2 weeks following gene delivery TGF-beta1 levels in joint fluid were significantly higher than the levels in the controls for 2 weeks.. Direct gene delivery into the joint cavity is feasible with the in vivo gene delivery method using adenovirus vector and would be clinically applicable.

Topics: Adenoviridae; Animals; Cartilage; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Guinea Pigs; Knee Joint; Lac Operon; Osteoarthritis; Synovial Membrane; Tissue Distribution; Transforming Growth Factor beta

To examine whether synoviocytes from patients with rheumatoid arthritis (RA) have a stronger growth ability than those from patients with osteoarthritis (OA), and to determine whether these synoviocytes clonally expand in situ.. Synovial tissues from 13 RA patients and 4 OA patients were cultured, and their ability to form colonies in soft agarose was examined. RA and OA synoviocytes were also examined in varying concentrations of fetal calf serum (FCS)-containing medium to test the effects of FCS on colony formation. DNA was extracted from clones with colony-forming ability in nonpannus lesions and from synoviocytes in pannus lesions. Restriction fragment length polymorphism (RFLP) analysis was used to examine phosphoglycerate kinase 1 (PGK-1) gene patterns. Production of cytokines by these cells was also assessed.. All 13 RA synoviocytes exhibited colony formation, whereas none of the 4 OA synoviocytes did. This tendency was also seen with all of the concentrations of FCS examined, although growth varied in a dose-dependent manner. In contrast to OA synovial clones, cloned RA synoviocytes obtained from colonies exhibited a partial RFLP PGK-1 gene pattern, suggesting that the clones originated from monoclonal cells. Of note, 3 of 7 noncloned synoviocytes from pannus lesions exhibited a monoclonal pattern. Pannus cells produced high levels of transforming growth factor beta and platelet-derived growth factor.. These findings suggest that synoviocytes with a strong growth ability are present in the rheumatoid synovium, and that these cells expand monoclonally, particularly in pannus lesions.

Topics: Adult; Aged; Arthritis, Rheumatoid; Cartilage; Cell Division; Clone Cells; Gene Expression Regulation, Enzymologic; Humans; Hyperplasia; Male; Middle Aged; Osteoarthritis; Phosphoglycerate Kinase; Platelet-Derived Growth Factor; Polymorphism, Restriction Fragment Length; Stem Cells; Synovial Membrane; Transforming Growth Factor beta

Parathyroid hormone-related protein (PTHrP) is a major, locally expressed regulator of growth cartilage chondrocyte proliferation, differentiation, synthetic function, and mineralization. Because mechanisms that limit cartilage chondrocytes from maturing and mineralizing are diminished in osteoarthritis (OA), we studied PTHrP expression by articular chondrocytes.. PTHrP was studied in normal knee cartilage samples and cultured articular chondrocytes, and in cartilage specimens from knees with advanced OA, obtained at the time of joint replacement.. PTHrP was more abundant in OA than in normal human knee articular cartilage. Both demonstrated PTH/PTHrP receptor expression. PTHrP 1-173, one of three alternatively spliced PTHrP isoforms, was exclusively expressed and induced by transforming growth factor beta in cultured chondrocytes. Chondrocytes mainly used the GC-rich P2 alternative promoter to express PTHrP messenger RNA. Inhibition by PTHrP 1-173, but not by PTHrP 1-146 or PTHrP 1-87, of inorganic pyrophosphate (PPi) elaboration suggested selective functional properties of the 1-173 isoform. Exposure to a neutralizing antibody to PTHrP increased PPi elaboration by articular chondrocytes.. Increased expression of PTHrP, including the 1-173 isoform, has the potential to contribute to the pathologic differentiated functions of chondrocytes, including mineralization, in OA.

Topics: Adult; Aged; Calcium Pyrophosphate; Cartilage, Articular; Extracellular Space; Humans; Middle Aged; Osteoarthritis; Parathyroid Hormone-Related Protein; Peptide Fragments; Promoter Regions, Genetic; Protein Isoforms; Proteins; Receptor, Parathyroid Hormone, Type 1; Receptors, Parathyroid Hormone; Transforming Growth Factor beta

One of the most prominent alterations that characterizes osteoarthritic cartilage damage is a reduction of proteoglycan content, reflecting an imbalance between synthesis and release of proteoglycans. Both synthesis and release depend on the activity of cartilage cells. Chondrocytes in the upper layer of moderately osteoarthritic human knee cartilage appear to be phenotypically altered, including diminished proteoglycan synthesis. Transforming growth factor-beta (TGF-beta) as a multifunctional growth factor has differential effects believed to depend on the differentiation stage of the target cells. We tested the effect of TGF-beta on phenotypically altered chondrocytes in osteoarthritic cartilage.. Human articular cartilage was cultured 4 days with or without TGF-beta. Proteoglycan synthesis was determined by (35)SO4(2-) incorporation (biochemically and by autoradiography) for the upper and deep layer separately.. Osteoarthritic cartilage proved more sensitive to TGF-beta than normal cartilage. Proteoglycan synthesis of osteoarthritic cartilage was stimulated significantly more by 5 ng/ml TGF-beta than normal cartilage. For normal cartilage this increase was equally divided among the upper and deeper layer of the explants. For osteoarthritic cartilage the increase in proteoglycan synthesis could largely be attributed to the upper layer. Autoradiography revealed that the relative (35)SO4(2-) incorporation in the cell clusters, present in the upper layer of osteoarthritic cartilage, was significantly increased upon the addition of TGF-beta.. Osteoarthritic cartilage is more sensitive to TGF-beta than normal cartilage because phenotypically changed chondrocytes in the damaged upper layer of osteoarthritic cartilage are more sensitive to TGF-beta than chondrocytes in the more intact deep layer and are more sensitive than the chondrocytes of normal cartilage. TGF-beta appears to redifferentiate the phenotypically altered chondrocytes in osteoarthritic cartilage.

Topics: Aged; Aged, 80 and over; Autoradiography; Cartilage; Cell Differentiation; Cells, Cultured; Female; Humans; Male; Osteoarthritis; Phenotype; Proteoglycans; Transforming Growth Factor beta

The transforming growth factor-beta 1 was known as having the most important influence on chondrocytes among various growth factors, being abundant in articular chondrocytes and osteocytes. We performed in vitro monolayer cultures of human articular chondrocytes from normal and osteoarthritic patients and studied the transforming growth factor-beta 1 responsiveness of those chondrocytes. The cell-growth curve indicated that the primary osteoarthritic chondrocyte culture with transforming growth factor-beta 1 showed a more rapid growth pattern than normal chondrocytes with or without TGF-beta 1 and osteoarthritic chondrocytes without TGF-beta 1. The osteoarthritic group showed a sharp decline in growth pattern with subsequent culture. The shape of osteoarthritic chondrocytes was bigger and more bizarre compared to those of normal chondrocytes. With subsequent culture, this change became prominent. The transforming growth factor-beta 1 increased the [3H]-TdR uptake in each group. The phenotypes of chondrocytes were more clearly expressed in the normal group. The chondrocytes lost their phenotype (production of collagen type II) following subculture in each group. The transforming growth factor-beta 1 could not inhibit or delay the dedifferentiation process (loss of phenotype).

Topics: Cartilage, Articular; Cell Division; Cells, Cultured; Humans; Osteoarthritis; Reference Values; Transforming Growth Factor beta

Osteoarthritic lesions were observed in the mandibular condyle cartilage of mice aged 7 months and older. These lesions appeared as fibrillations along the articular surface and were accompanied by reduced extracellular matrix synthesis and chondrocyte proliferation. Explants of mandibular condyle cartilage were cultured in serum-free BGJb medium supplemented with ascorbic acid (300 micrograms/ml), penicillin (100 U/ml) and streptomycin (100 micrograms/ml) for up to 72 h. Cultures were further supplemented with either hTGF-beta 1 (0.1-5.0 ng/ml) or human IL-1 alpha (40 U/ml). [3H]thymidine (2 microCi/ml) and [35S]SO4 (10 microCi/ml) were added to the culture medium for the last 24 h of culture and incorporation into DNA and sulfated proteoglycans, respectively, studied. The results indicated that protein and DNA contents, [3H]thymidine and [35S]SO4 incorporation, as well as the specific activity of alkaline phosphatase, were increased by TGF-beta 1. Addition of 1.0 or 5.0 ng/ml hTGF-beta 1 to the cultures for 48 h, had the most stimulatory effect on matrix synthesis and cell proliferation, whereas 0.1 ng/ml hTGF-beta 1 appeared to be inhibitory when compared to controls. Increased [35S]SO4 labeling of chondrocyte clusters was observed by autoradiography in tissue sections from cultures treated with TGF-beta 1 (1.0 and 5.0 ng/ml). In contrast, IL-1 alpha exerted inhibitory effects on cell proliferation and matrix synthesis. However, it induced the activity of acid phosphatase in these cultures. The results of the present study show that IL-1 alpha had catabolic effect on his tissue, while TGF-beta 1 enhanced proliferation and induced synthetic activity of the cartilage cells. Such action by TGF-beta suggests the existance of a possible repair process in osteoarthritic cartilage of the temporo-mandibular joint of aged mice.

Topics: Aging; Animals; Cartilage, Articular; DNA; Extracellular Matrix; Female; Humans; Interleukin-1; Mandibular Condyle; Mice; Mice, Inbred ICR; Osteoarthritis; Proteins; Temporomandibular Joint; Temporomandibular Joint Disorders; Transforming Growth Factor beta

The temporo-mandibular joint of aged mice develops osteoarthritic (OA) degenerative lesions. Adult chondrocytes have a low rate of cell replication, and cartilage repair potential is very limited. One of the major problems in OA is the low rate of matrix synthesis and the inability of the chondrocytes to exceed the rate of matrix degradation. These combined factors lead to the overall destruction of the cartilage as seen in OA. Cartilage degradation is mediated by elevated proteolytic activity of enzymes. Among the enzymes degrading cartilage are the metalloproteinases, stromelysin and collagenase. Other proteinases that may potentially participate in matrix degradation are the lysosomal enzymes cathepsin B, D, and L, and acid phosphatase. On the other hand, alkaline phosphatase (ALP) is an enzyme that has been shown to be a marker for anabolic activity in skeletal tissues such as bone and cartilage. The cartilage of the mandibular condyle in the T-M-J from aged mice reveals OA lesions. An overall reduction of cell proliferation and sulfated proteoglycan synthesis has been also shown in this joint. In the present study the effects of hTGF-beta on the stimulation of DNA and sulfated GAG synthesis and ALP activity were studied. Mandibular condyle cartilage obtained from 12-month-old ICR male mice were cultured in BGJb serum-free medium for 24-72 hours, supplemented with 0.1-10 ng/ml hTGF-beta 1. 3H-thymidine and 35S-sulfate were added for the last 24 hours of the culture and their incorporation into DNA and sulfated GAGs respectively, as well as the activity of ALP, were determined. Results indicated that hTGF-beta 1 enhanced the incorporation of both 3H-thymidine and of 35S-sulfate into cartilage cultures of aged mice, and also induced ALP activity. It thus appeared that in OA degenerating articular cartilage, the chondrocytes could be stimulated in vitro to proliferate and to synthesize new matrix, thus indicating induced anabolic activity in the tissue.

Topics: Alkaline Phosphatase; Animals; Cartilage, Articular; Cell Division; Culture Techniques; Extracellular Matrix; Glycosaminoglycans; Histocytochemistry; Male; Mice; Microscopy, Electron; Osteoarthritis; Proteoglycans; Temporomandibular Joint; Transforming Growth Factor beta

To examine, by immunohistochemistry, the localization and distribution of human collagenase-3 in normal, osteoarthritis (OA), and rheumatoid arthritis (RA) cartilage, and to investigate the effects of interleukin-1beta (IL-1beta) and transforming growth factor beta (TGFbeta) on the synthesis and distribution of collagenase-3.. Human cartilage specimens were obtained from tibial plateaus. In the first series of experiments, the OA specimens were excised from fibrillated and nonfibrillated areas of cartilage, and RA specimens were excised from lesional areas, including the cartilage-pannus junction when present. In the second series, full strips of cartilage were processed for culture in the presence or absence of IL-1beta (100 units/ml) or TGFbeta (150 ng/ml). Each specimen was processed for immunohistochemical analysis using a collagenase-3 monoclonal antibody.. The number of cells that stained for collagenase-3 in normal cartilage was very low (approximately 3%). In OA cartilage, the percentage increased dramatically, and no difference was found between fibrillated and nonfibrillated areas. A statistically significant increase in the percentage of cells staining for collagenase-3 was found in the deep layer compared with the superficial layer. This finding was noted in both the fibrillated areas (mean +/- SEM 58.4 +/- 1.6% and 40.1 +/- 3.9%, respectively; P < 0.007) and the nonfibrillated areas (55.4 +/- 3.2% and 43.2 +/- 2.7%; P < 0.01). Similarly, RA cartilage showed a statistically significant (P < 0.001) increase in the level of chondrocytes staining positive for collagenase-3 in the deep layers (46.4 +/- 4.1%) compared with the superficial layers (26.2 +/- 3.4%). In these RA specimens, the numbers of positively staining chondrocytes were similar both close to and at a distance from the pannus junction. Both IL-1beta and TGFbeta increased the number of chondrocytes producing collagenase-3. Interestingly, in normal specimens, TGFbeta had a predominant effect in the deep layers, while IL-1beta had a greater effect on the superficial layers.. This study demonstrates that, in situ, the increase in the level of chondrocytes synthesizing collagenase-3 in arthritic cartilage is predominant in the deep layers. The results further indicate that TGFbeta can up-regulate the level of this enzyme and, in normal cartilage in vitro, can cause a mimicking of the in situ distribution observed in arthritic cartilage.

Topics: Aged; Arthritis, Rheumatoid; Cartilage, Articular; Cell Count; Cells, Cultured; Collagenases; Humans; Immunohistochemistry; Interleukin-1; Matrix Metalloproteinase 13; Middle Aged; Osteoarthritis; Transforming Growth Factor beta; Up-Regulation

Members of the TGF-beta superfamily are important regulators of skeletal development. TGF-betas signal through heteromeric type I and type II receptor serine/threonine kinases. When over-expressed, a cytoplasmically truncated type II receptor can compete with the endogenous receptors for complex formation, thereby acting as a dominant-negative mutant (DNIIR). To determine the role of TGF-betas in the development and maintenance of the skeleton, we have generated transgenic mice (MT-DNIIR-4 and -27) that express the DNIIR in skeletal tissue. DNIIR mRNA expression was localized to the periosteum/perichondrium, syno-vium, and articular cartilage. Lower levels of DNIIR mRNA were detected in growth plate cartilage. Transgenic mice frequently showed bifurcation of the xiphoid process and sternum. They also developed progressive skeletal degeneration, resulting by 4 to 8 mo of age in kyphoscoliosis and stiff and torqued joints. The histology of affected joints strongly resembled human osteo-arthritis. The articular surface was replaced by bone or hypertrophic cartilage as judged by the expression of type X collagen, a marker of hypertrophic cartilage normally absent from articular cartilage. The synovium was hyperplastic, and cartilaginous metaplasia was observed in the joint space. We then tested the hypothesis that TGF-beta is required for normal differentiation of cartilage in vivo. By 4 and 8 wk of age, the level of type X collagen was increased in growth plate cartilage of transgenic mice relative to wild-type controls. Less proteoglycan staining was detected in the growth plate and articular cartilage matrix of transgenic mice. Mice that express DNIIR in skeletal tissue also demonstrated increased Indian hedgehog (IHH) expression. IHH is a secreted protein that is expressed in chondrocytes that are committed to becoming hypertrophic. It is thought to be involved in a feedback loop that signals through the periosteum/ perichondrium to inhibit cartilage differentiation. The data suggest that TGF-beta may be critical for multifaceted maintenance of synovial joints. Loss of responsiveness to TGF-beta promotes chondrocyte terminal differentiation and results in development of degenerative joint disease resembling osteoarthritis in humans.

Topics: Animals; Bone and Bones; Cartilage, Articular; Cell Differentiation; Gene Expression; Growth Plate; Hedgehog Proteins; Humans; Hypertrophy; Joints; Mice; Mice, Transgenic; Osteoarthritis; Polymerase Chain Reaction; Protein Biosynthesis; Protein Serine-Threonine Kinases; Receptor Protein-Tyrosine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; RNA, Messenger; Synovial Membrane; Trans-Activators; Transcription, Genetic; Transforming Growth Factor beta

Since many cytokines have been identified in chronically inflamed human synovium, it is possible that particular cytokines or combinations of cytokines play dominant roles in driving or inhibiting metabolic processes important to inflammation. To assess these possibilities, we compared selected effects of individual cytokines and their binary, ternary, and higher combinations in human synovial cell cultures.. Cytokines studied known to occur in human synovial tissue included: interleukin 1beta (IL-1beta), IL-6, tumor necrosis factor-alpha, granulocyte macrophage colony stimulating factor, interferon-gamma, acidic fibroblast growth factor (aFGF), basic FGF (bFGF), platelet derived growth factor, transforming growth factor-beta1, connecting tissue activating peptide-III, and epidermal growth factor. The growth related effects of these agents singly and in combinations were assessed by measuring newly synthesized [3H]DNA and [14C]GAG (glycosaminoglycan) in human synovial cell cultures. Cytokine induced synthesis of prostaglandin E2 (PGE2) was measured by ELISA.. Most cytokine combinations resulted in additive/synergistic anabolic effects, except when IL-1beta was present; IL-1beta was markedly antagonistic to the mitogenic effects of other cytokines tested. Combinations of platelet derived cytokines were the most potent stimulators of DNA synthesis, while combinations of synovial derived cytokines were more active in stimulating GAG synthesis. Synovial cells exposed simultaneously to both platelet and synovial derived cytokines produced large quantities of [14C]GAG and showed a modest increase in [3H]DNA synthesis. IL-1beta, alone or in combinations, was dominant with respect to stimulation of PGE2 synthesis. Acetylsalicylic acid substantially interfered with all the effects of cytokine combinations measured.. Quantitative alterations in synovial cell synthesis of GAG and DNA varied greatly depending on the ambient mixture of cytokines. Virtually all combinations of cytokines tested gave rise to large increases in synovial cell synthesis of GAG. Four platelet derived cytokines, a "physiologic combination," appeared to be dominant agents in stimulating DNA synthesis. This effect was profoundly reduced by the antagonistic effect of IL-1beta, mediated in part by PGE2. The patterns of cytokine combination induced metabolic effects suggest that the "cytokine network" has a significant measure of redundancy with respect to control of synovial cell metabolism.

Topics: Arthritis, Rheumatoid; Aspirin; Cells, Cultured; Connective Tissue; Culture Media, Conditioned; Cytokines; Dinoprostone; DNA; Drug Synergism; Epidermal Growth Factor; Fibroblast Growth Factors; Glycosaminoglycans; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Hydrocortisone; Inflammation; Insulin-Like Growth Factor I; Interferon-gamma; Interleukin-6; Osteoarthritis; Peptides; Platelet-Derived Growth Factor; Recombinant Proteins; Synovial Membrane; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

To discover if alpha smooth muscle actin expression and myofibroblastic differentiation are induced in synovial fibroblasts by cytokines found in the inflamed RA joint.. Immunofluorescent microscopy and western blotting were used to examine different cultures of human synovial fibroblasts for expression of alpha actin in the presence of the cytokines transforming growth factor beta (TGF beta 1), interleukin 1 alpha (IL1 alpha), IL4, IL6, tumour necrosis factor alpha (TNF alpha), and basic fibroblast growth factor (FGF).. A small but significant population of cells (14.4 +/- 12.9%) expressed alpha actin under standard culture conditions. Upon treatment with TGF beta 1 there was a pronounced increase in the number of cells expressing alpha actin (68.1 +/- 5.49%), accompanied by a change in morphology to a myofibroblast-like phenotype. Other cytokines found within the inflamed joint such as IL1, TNF alpha, IL6, and basic FGF failed to induce alpha actin expression. However, IL4, which is normally absent or only present at low concentrations in the RA joint had a similar effect to TGF beta 1. It was also found that basic FGF inhibited the induction of alpha actin expression by TGF beta 1 and IL4.. In the presence of TGF beta 1 or IL4, fibroblasts derived from synovial tissue or synovial fluid are induced to differentiate into myofibroblast-like cells containing the alpha smooth muscle form of actin. This differentiation is inhibited by basic FGF. It is suggested that the balance between these particular cytokines may be important in the modulation of fibroblast behaviour, which could have significant effects on joint repair mechanisms and the generation of fibrous tissue within the rheumatoid joint.

Topics: Actins; Arthritis; Arthritis, Rheumatoid; Blotting, Western; Cell Differentiation; Cells, Cultured; Fibroblast Growth Factor 2; Fibroblasts; Humans; Interleukin-4; Microscopy, Fluorescence; Osteoarthritis; Synovial Membrane; Transforming Growth Factor beta

Eighty-five percent of male STR/ort mice develop osteoarthritic lesions of the knee joint by 35 weeks of age. We have developed a non-radioactive in-situ hybridization method using digoxigenin-labeled oligonucleotide probes to study the expression of the cytokines interleukin (IL) 1 alpha, Il-1 beta and IL-6 and the growth factors insulin-like growth factor-1 (IGF-1) and transforming growth factor beta (TGF beta 1) during the development of osteoarthritis (OA) in this model. Age- and sex-matched CBA mice, which do not develop OA, showed no detectable expression of any of the cytokines or growth factors studied. In contrast, 20-week-old STR/ort mice with no OA lesions showed positive expression [positive: (+)] for all the cytokines and growth factors studied. At 35 weeks of age, STR/ort mice with varying grades of OA showed positive (+) or strong (++) signals for both cytokines and growth factors throughout the tibial articular cartilage. The strongest signal was seen in areas where OA lesions were present. In areas of histologically-normal cartilage adjacent to the lesions, the signals were still positive but weaker. Fifty-week-old STR/ort mice with OA lesions showed a similar pattern of expression to 35-week-old mice. Thirty-five or 50-week-old STR/ort mice with no OA lesions had much reduced expression compared with those with OA lesions. These mice may be indicative of those STR/ort mice which do not develop OA. The results seen in the STR/ort together with previous biochemical analyses are consistent with an up-regulation of anabolic growth factors and catabolic cytokines in the prelesional stages of OA with anabolic effects predominating. At later stages of OA, the effects of catabolic factors appear to predominate and osteoarthritic lesions become evident.

Topics: Aging; Animals; Cartilage, Articular; Chondrocytes; Growth Substances; In Situ Hybridization; Insulin-Like Growth Factor I; Interleukins; Knee Joint; Male; Mice; Mice, Inbred CBA; Mice, Inbred Strains; Osteoarthritis; Transforming Growth Factor beta

This study tested the effect of fluid-induced shear on interleukin-6 expression in normal human articular chondrocytes in vitro. As determined by Northern blot analysis, interleukin-6 mRNA expression occurs in chondrocytes from osteoarthritic cartilage but not in normal chondrocytes. Applying fluid-induced shear stress to primary high density cultures of chondrocytes increased interleukin-6 mRNA signal 4-fold at 1 hour and 10 to 15-fold at 48 hours compared with unsheared control cultures. At 48 hours, fluid-induced shear stress increased interleukin-6 protein levels in the culture medium 9 to 10-fold compared with unsheared controls. mRNA signals for interleukin-1alpha, interleukin-1beta, and tumor necrosis factor-alpha in RNA from sheared or control chondrocytes were not detected by Northern blotting. Transforming growth factor-beta mRNA signal was detectable but was not affected by shear. In contrast, human lung fibroblasts (WI-38) responded to fluid-induced shear with increased signal for transforming growth factor-beta, but not interleukin-6, mRNA. Both cell types did respond to interleukin-1alpha with increased interleukin-6 mRNA signal. These data demonstrated that distortional forces, such as fluid-induced shear stress, alter interleukin-6 levels in normal chondrocytes in vitro and suggest that increased interleukin-6 expression in osteoarthritic cartilage may result, in part, from alterations in the mechanical loading of the tissue.

Topics: Adult; Aged; Blotting, Northern; Cartilage, Articular; Cells, Cultured; Child, Preschool; Female; Fibroblasts; Humans; Interleukin-1; Interleukin-6; Lung; Male; Middle Aged; Osteoarthritis; Reference Values; RNA, Messenger; Stress, Mechanical; Time Factors; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Tissue inhibitors of metalloproteinases (TIMPs) inhibit the extracellular matrix (ECM) metalloproteinases (MMPs). To determine the source of TIMPs in synovial fluids of patients with osteoarthritis (OA), the ability of chondrocytes to express TIMP-2 and its regulation by agents found in inflammed joints was investigated. The constitutive TIMP-2 mRNA expression was demonstrated in chondrocytes from normal bovine, human OA and normal cartilage. The cross-hybridization of human and bovine TIMP-2 suggested its evolutionary conservation. Serum, IL-1, IL-6 and TGC-beta were unable to augment considerably the basal expression of TIMP-2 mRNA. TIMP-1 RNA expression in chondrocytes from human OA cartilage was elevated compared to non-OA chondrocytes, while TIMP-2 mRNA levels were similar in both. IL-1 beta, IL-6 and TGF-beta did not affect TIMP-2 expression but TGF-beta induced TIMP-1 mRNA in human OA chondrocytes. TIMP-2 and TIMP-1 are therefore differentially regulated in chondrocytes and the basal TIMP-2 levels may be needed for the cartilage ECM integrity.

Topics: Animals; Cartilage, Articular; Cattle; Cells, Cultured; Cytokines; Glycoproteins; Growth Substances; Humans; Interleukins; Matrix Metalloproteinase Inhibitors; Osteoarthritis; Protease Inhibitors; Proteins; Reference Values; RNA, Messenger; Tissue Inhibitor of Metalloproteinase-2; Tissue Inhibitor of Metalloproteinases; Transforming Growth Factor beta

The age-related expression of the extracellular matrices in human mandibular condyle was examined.. The distribution patterns of types I to V collagens, laminin, fibronectin, fibronectin receptor, and transforming growth factor beta in 34 human mandibular condyles dissected from autopsy specimens were studied by immunohistochemical procedure with special attention on the age-related changes.. Type I collagen was detected in the full layer of the condylar cartilage, and a stronger immunoreaction was delineated in the articular and cartilage zone. Types II and III collagen were mainly localized in the fibrocartilage zone. Type IV collagen and laminin were detected not only in the basement membrane of the blood vessels but also in the degenerated lesion where the expression of transforming growth factor beta was also detected. Immunostaining of type V collagen and fibronectin was noted in the perichondrocytic area, whereas that of fibronectin receptor was seen in the chondrocytes. In materials from younger cadavers types I, II, IV and V collagens, fibronectin, its receptor, and laminin showed stronger expression in the degenerative lesions than in the normal portions. In the sections from cadavers over the seventh decade, the immunoreaction of extracellular matrices was weak compared with the younger materials, and no increased reaction of extracellular matrices in the degenerative lesions was detected. In addition, severe osteoarthrosis was frequently seen in the older materials in macroscopic findings.. These results suggest that the expression of extracellular matrices thus seems to be closely related to aging and degenerative changes in the condyle.

Topics: Adolescent; Adult; Age Factors; Aged; Aged, 80 and over; Aging; Cartilage, Articular; Collagen; Extracellular Matrix; Extracellular Matrix Proteins; Female; Fibronectins; Humans; Immunoenzyme Techniques; Laminin; Male; Mandibular Condyle; Middle Aged; Osteoarthritis; Receptors, Fibronectin; Transforming Growth Factor beta

Physiological and pathological degradation of cartilage extracellular matrix (ECM) is regulated by the balance between tissue inhibitors of metalloproteinases (TIMPs) and matrix metalloproteinases (MMPs). We examined the potential of chondrocytes from normal bovine or human osteoarthritic (OA) cartilage to express RNA for the new inhibitor TIMP-3 and studied its regulation by an inducer of matrix synthesis, transforming growth factor-beta (TGF-beta). Freshly released chondrocytes constitutively expressed three transcripts of TIMP-3 that are induced by serum factors. In primary cultures of chondrocytes, one of these factors, TGF-beta, increased TIMP-3 mRNA in a dose-dependent fashion that required de novo protein synthesis and transcription. TGF-beta did not alter stability of the TIMP-3 transcripts in RNA decay time-courses, suggesting a transcriptional control. Nuclear run-on assays confirmed increased rate of TIMP-3 gene transcription by TGF-beta. An antiinflammatory glucocorticoid, dexamethasone, inhibited the basal, and suppressed partially the TGF-beta-inducible, TIMP-3 expression in primary bovine and human chondrocytes. DNA sequencing of bovine TIMP-3 cDNA revealed an open reading frame of a 211-amino-acid protein containing signal peptide and 12 conserved cysteines. The encoded protein differed from human TIMP-3 at four positions. The constitutive expression and evolutionary conservation of TIMP-3 imply its important function. TIMP-3 induction by TGF-beta suggests the role of this factor and TIMP-3 in cartilage remodeling with important implications for arthritis.

Topics: Amino Acid Sequence; Animals; Base Sequence; Blood; Cartilage, Articular; Cattle; Cells, Cultured; Cloning, Molecular; Dexamethasone; DNA, Complementary; Gene Expression Regulation; Glucocorticoids; Humans; Molecular Sequence Data; Osteoarthritis; Protease Inhibitors; Protein Biosynthesis; Protein Sorting Signals; Proteins; RNA, Messenger; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Tissue Inhibitor of Metalloproteinase-3; Transcription, Genetic; Transforming Growth Factor beta

Classically, osteoarthritis (OA) has been considered a noninflammatory disease. However, the detection of selected inflammatory mediators in osteoarthritic fluid, in the absence of significant inflammatory cell infiltrate, is increasingly appreciated. We sought to identify the inflammatory component in human OA-affected cartilage that may be involved in cartilage damage/destruction. Using Western blot analysis and an antibody to the conserved region of nitric oxide synthase (NOS), we have observed up-regulation of NOS, one of the "key players" of inflammation, in chondrocytes of OA-affected patients. Remarkably, none of the cartilage samples examined from normal joints demonstrated detectable amounts of this NOS. Western blot analysis using the same alpha-NOS antibody indicated that this NOS from OA-affected cartilage (OA-NOS) was larger in size than (and distinct from) transfected human hepatocyte or murine inducible NOS (iNOS) (150 versus 133 kD) and similar in size to neuronal constitutive NOS (ncNOS). Antibodies specific for iNOS showed binding to murine and human iNOS but not to OA-NOS, endothelial constitutive NOS, or ncNOS. Antibodies specific for ncNOS bound to ncNOS and also to OA-NOS, but not to murine or human iNOS or endothelial constitutive NOS. Incubation of OA cartilage in serum-free medium resulted in spontaneous release, for up to 72 h, of substantial amounts of nitrite (up to approximately 80 microM/100 mg wet tissue), which could be inhibited by at least 80% with various inhibitors of iNOS, including inhibitors of protein synthesis and transcription factor NF-kappa B, but which (unlike murine macrophage iNOS) was not sensitive to hydrocortisone or TGF-beta. Exposure of OA-affected cartilage to interleukin 1 beta, tumor necrosis factor-alpha, and lipopolysaccharide resulted in approximately 20-50% augmentation of nitrite accumulation, which was also sensitive to cycloheximide and pyrrolidine dithiocarbamate. Hence, our data indicate that OA-NOS (based on immunoreactivity and molecular weight) is similar to ncNOS and that it releases nitric oxide, which may contribute to the inflammation and pathogenesis of cartilage destruction in OA.

Topics: Animals; Anti-Inflammatory Agents; Cartilage; Cattle; Cells, Cultured; Enzyme Induction; Humans; Hydrocortisone; Neurons; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase; Osteoarthritis; Transforming Growth Factor beta; Up-Regulation

Fibronectin is a glycoprotein involved in cell matrix interactions. In osteoarthritis, fibronectin levels in the lesion cartilage are elevated up to 20-fold above control levels. In these experiments, explants of disease-free cartilage cultured in the presence of a combination of TGF beta 1 and the sulfated fucopolysaccharide, fucoidan, accumulated fibronectin at levels comparable to those found in osteoarthritic lesions. TGF beta 1 increased fibronectin synthesis, most of which was released to the medium. The addition of fucoidan favored retention of the newly synthesized fibronectin within the matrix. The fibronectin which accumulated as a result of these treatments was similar to the fibronectin in normal and osteoarthritic cartilage with respect to the ED-B+ alternative splice form. No change in the proteoglycan content of the cartilage explants with elevated fibronectin levels was detected.

Topics: Animals; Cartilage, Articular; Cells, Cultured; Dogs; Fibronectins; Glycosaminoglycans; Hip Dislocation, Congenital; In Vitro Techniques; Osteoarthritis; Pentosan Sulfuric Polyester; Polysaccharides; Proteoglycans; Transforming Growth Factor beta

The ingress of inflammatory cells into the rheumatoid (RA) synovial tissue (ST) plays a role in the pathogenesis of this disease. Transforming growth factor beta (TGF-beta) may play a role in this process. We have investigated the distribution of endoglin, a newly described receptor for TGF-beta 1 and -beta 3, in RA compared to osteoarthritis (OA) or normal ST. Immunohistochemical analysis was carried out using an anti-TGF-beta 1 monoclonal antibody (mAb) as well as 10 mAbs raised against various epitopes of endoglin. This study was performed on ST from 10 patients with RA, 10 with OA, and 4 normal individuals. TGF-beta 1 expression was significantly up-regulated on RA compared to OA and normal ST lining cells, interstitial macrophages, and endothelial cells (P < 0.05). All anti-endoglin mAbs uniformly reacted with endothelial cells in RA, OA, and normal STs. However, 3 out of 10 anti-endoglin mAbs reacted with significantly more RA versus normal ST lining cells (P < 0.05), as well as RA compared to OA and normal macrophages (P < 0.05). There was a positive correlation between TGF-beta 1 and endoglin reactivity on the synovial lining layer and subsynovial macrophages (P < 0.05). These results indicate that TGF-beta 1 and certain epitopes of endoglin, a TGF-beta 1 and -beta 3 receptor, are up-regulated on myeloid elements in RA compared to normal ST. Endoglin is also present on ST endothelia, and its expression may also be increased on OA compared to normal ST lining cells. These findings implicate endoglin in the pathogenesis of RA.

Topics: Antibodies, Monoclonal; Antigens, CD; Arthritis, Rheumatoid; Endoglin; Humans; Membrane Glycoproteins; Osteoarthritis; Receptors, Cell Surface; Synovial Membrane; Transforming Growth Factor beta; Vascular Cell Adhesion Molecule-1

Regulation of chondrocyte secretory functions and proliferation by cytokines and growth factors is central to cartilage development and maintenance of homeostasis in the mature organism. Depending on the type of extracellular stimulus, chondrocytes can be induced to enter a catabolic matrix degrading or anabolic matrix forming functional program. Interleukin I and transforming growth factor beta are the prototypic stimuli for the catabolic and anabolic program, respectively. Insight into the regulation of chondrocytes by cytokines and growth factors provides the basis for improved concepts of osteoarthritis pathogenesis and new perspectives for therapeutic interventions.

Topics: Cartilage; Cells, Cultured; Cytokines; Humans; Interferon-gamma; Interleukin-1; Interleukin-6; Osteoarthritis; Signal Transduction; Transforming Growth Factor beta

Osteoarthritis (OA) is characterized by focal cartilage destruction and osteophyte formation. Triple injections of transforming growth factor beta (TGF beta) in normal murine knee joints revealed that this growth factor can elicit osteophytes at sites characteristic for OA. Moreover, enhanced cartilage proteoglycan synthesis is induced and cartilage proteoglycan content is increased. Exuberant repair reactions may be pathogenic and indeed focal cartilage proteoglycan loss and disorganized chondrocyte spacing is noted in the tibial plateau after 1 month. Although TGF beta is immunosuppressive and can counteract interleukin 1 induced cartilage damage, suggesting potential therapeutic application in joint inflammation, it should probably be viewed as a pathogenic factor in OA.

Topics: Animals; Cartilage, Articular; Disease Models, Animal; Injections, Intra-Articular; Insulin-Like Growth Factor I; Knee Joint; Mice; Osteoarthritis; Proteoglycans; Transforming Growth Factor beta

This study evaluated the effects of basic fibroblast growth factor, transforming growth factor-beta 1, insulin-like growth factor-1, and insulin on the incorporation of thymidine and sulfate in human osteoarthritic articular cartilage. Tissue explants were obtained from 11 patients undergoing total knee arthroplasty and were categorized as nonfibrillated or fibrillated cartilage. The explants were cultured for 22 days, with changes of medium and growth factor every 72 hours, and labeled with [3H]thymidine and [35S]sulfate. Growth factors were used in the following concentrations: basic fibroblast growth factor at 1, 10, and 100 ng/ml; transforming growth factor-beta 1 at 0.5, 5, and 50 ng/ml; insulin-like growth factor-1 at 0.15, 1.5, and 15 ng/ml; and insulin at 0.05, 0.5, and 5 micrograms/ml. Basic fibroblast growth factor decreased thymidine incorporation to 70% and sulfate incorporation to less than 20% that of the growth factor-free controls. Transforming growth factor-beta 1 had no significant effect on thymidine incorporation, whereas the concentrations studied inhibited sulfate incorporation to approximately 40% that of the controls. At the concentrations tested, insulin-like growth factor-1 had no significant effect on incorporation of either thymidine or sulfate. In contrast, insulin significantly stimulated the incorporation of both. Compared with growth factor-free controls, insulin maximally increased thymidine incorporation by a factor (+/- SEM) of 2.36 +/- 0.47 and 1.69 +/- 0.19 in nonfibrillated and fibrillated explants, respectively; sulfate incorporation was maximally increased 1.60 +/- 0.24 and 1.92 +/- 0.29-fold for nonfibrillated and fibrillated explants, respectively. Of the factors tested, insulin demonstrated the greatest promise for promoting a synthetic response that may contribute to the regeneration of osteoarthritic cartilage.

Topics: Aged; Aged, 80 and over; Cartilage, Articular; Cell Division; Culture Techniques; DNA; Extracellular Matrix; Fibroblast Growth Factor 2; Growth Substances; Humans; Insulin; Insulin-Like Growth Factor I; Knee Joint; Middle Aged; Osteoarthritis; Proteoglycans; Regeneration; Sulfates; Thymidine; Transforming Growth Factor beta

The ability of human transforming growth factor-beta 1 (hTGF-beta 1) to induce proliferation and matrix synthesis in articular cartilage of aging mice was studied using an organ culture system of mandibular condylar cartilage. An increased incorporation of 3H-thymidine into DNA and of 35S-SO4 into sulfated glycosaminoglycans was observed in cultures supplemented with 1-10 ng/ml TGF-beta and with 1% fetal calf serum. In these cultures numerous clusters of chondrocytes containing a well-developed rough endoplasmic reticulum were seen. Articular cartilage from mature animals often develops osteoarthritic degenerative lesions, thus the induction of cell proliferation and of matrix synthesis may be an indication of a tissue repair process in cartilage from maturing animals.

Topics: Aging; Animals; Cartilage, Articular; Cell Division; DNA; Glycosaminoglycans; Humans; Male; Mice; Mice, Inbred ICR; Microscopy, Electron; Organ Culture Techniques; Osteoarthritis; Transforming Growth Factor beta

Using in situ hybridization, we investigated the expression of mRNA for interleukin-1 beta (IL1 beta), interleukin-6 (IL6), and transforming growth factor-beta-1 (TGF beta 1) in sections of developing bone in human osteophytes. The expression was related to the cellular activity of alkaline phosphatase to aid in the identification of pre-osteoblast populations. IL1 beta mRNA was localized in active osteoblasts within distinct areas of intramembranous ossification. However, the expression was sporadic and appeared to occur at a specific stage of the osteoblast life cycle. There was no IL1 beta mRNA expression in any cell types during endochondral ossification. IL6 mRNA expression was located within pre-osteoblasts and in newly differentiated and matrix-secreting osteoblasts; expression was absent or reduced in flattened, inactive osteoblasts. Weak or no IL6 expression was observed in chondroblasts and chondrocytes, respectively. However, there was a close association between IL6 mRNA expression and the differentiation of mesenchymal cells into osteoblasts. TGF beta 1 expression was localized to osteoblasts apposed to bone or cartilage matrix; the intensity of expression correlated with matrix secretion. Chondroblasts and chondrocytes expressed lower but significant levels of TGF beta 1 mRNA; the expression was lost with the progression to calcifying cartilage. The three cytokines studied were differentially expressed both temporally and spatially, suggesting different roles for each in osteoblast and chondrocyte function.

Topics: Alkaline Phosphatase; Biomarkers; Bone and Bones; Cartilage, Articular; Femur; Gene Expression; Hip Prosthesis; Humans; In Situ Hybridization; Interleukin-1; Interleukin-6; Osteoarthritis; Osteoblasts; RNA Probes; RNA, Messenger; Transforming Growth Factor beta

To investigate whether growth factors stored in bone might explain the increased bone density and resistance to osteoporosis in generalized osteoarthritis.. Levels of insulin-like growth factor (IGF) types I and II and transforming growth factor beta (TGF beta) were measured in extracts of cortical bone from the iliac crest obtained at necropsy from subjects with or without osteoarthritis of the hands.. Concentrations of IGF-I, IGF-II, and TGF beta were significantly higher in extracts of bone powder from subjects in the osteoarthritis group than in extracts from subjects in the control group.. The results suggest that the increased bone density and resistance to osteoporosis in patients with osteoarthritis may be associated with increased skeletal concentrations of IGF-I, IGF-II, and TGF beta and may reflect a generally increased biosynthetic activity of osteoblasts in these patients.

Topics: Aged; Bone Density; Female; Hand; Humans; Ilium; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Osteoarthritis; Osteoporosis; Radiography; Transforming Growth Factor beta

Osteoarthritis is a degenerative joint disease, characterized by the destruction of the articular cartilage. One of the first changes in the osteoarthritic articular cartilage is a reduction in proteoglycan content. In this study we demonstrate that transforming growth factor beta (TGF beta), a multi-functional growth factor, stimulates the proteoglycan synthesis of explants from human articular knee cartilage dose-dependently in vitro. Osteoarthritic cartilage proved to be much more sensitive to stimulation by TGF beta than normal healthy cartilage. This may indicate that TGF beta plays an important role in the repair of osteoarthritic cartilage.

Topics: Aged; Cartilage; Dose-Response Relationship, Drug; Female; Humans; Knee Joint; Male; Middle Aged; Osteoarthritis; Proteoglycans; Reference Values; Transforming Growth Factor beta

Transforming growth factor (TGF)-beta has been shown to promote tissue repair and have immunosuppressive actions, and has been proposed to have a role in rheumatoid arthritis (RA). Using immunohistochemical techniques with rabbit F(ab')2 antibodies raised against recombinant human TGF-beta 1, we have detected TGF-beta 1 in the synovial tissue and cartilage/pannus junction (CPJ) from 18/18 patients with RA. TGF-beta 1 was found predominantly in the thickened synovial lining layer in RA, but also detected in a perivascular pattern in the synovial interstitium as well as in occasional cells in the lymphoid aggregates. At the CPJ it was found both in cells at the distinct junction as well as in the transitional region of the diffuse fibroblastic zone. The cells staining for TGF-beta 1 were identified by double immunofluorescence staining as being from the monocyte/macrophage series as well as the type B synovial lining cells. TGF-beta 1 was also detected in the synovial membrane sections from 4/4 patients with systemic lupus erythematosus/mixed connective tissue disease and 5/8 patients with osteoarthritis, in a similar distribution to that seen in RA, and in the lining layer of 1/7 normal synovial membranes. These results add to histological evidence confirming that TGF-beta 1 is present in RA synovial cells and those from other arthritides. The distributions of TGF-beta 1 in RA synovial membrane reflects its known actions, as it can be detected at the CPJ, where it could induce repair, and close to activated cells upon which it may exert an immunosuppressive action.

Topics: Antibodies, Monoclonal; Arthritis, Rheumatoid; Dendritic Cells; Fibroblasts; Fluorescent Antibody Technique; Humans; Joints; Macrophages; Mixed Connective Tissue Disease; Osteoarthritis; Phenotype; Synovial Membrane; T-Lymphocytes; Transforming Growth Factor beta

Conditioned media obtained from fibroblasts cultured from rheumatoid and certain other inflammatory synovia were observed to stimulate [3H]thymidine incorporation in an indicator murine fibroblast line. Synovial fibroblasts derived from the joints of patients with osteoarthritis did not display this property. This effect persisted in culture for many weeks and occurred in the absence of co-stimulatory immune cells. Antibody neutralization studies implicated a role for basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF-beta), granulocyte/macrophage colony-stimulating factor (GM-CSF), and interleukin 1 beta (IL-1 beta) in the increased proliferative activity of synovial fibroblast-conditioned media. Synovial cell synthesis of bFGF, TGF beta 1, GM-CSF, IL-1 beta, and IL-6 was confirmed by 35S-methionine labeling and immunoprecipitation. The constitutive production of inflammatory and mitogenic cytokines by synovial fibroblasts may represent the result of long-term, phenotypic changes that occurred in vivo. Persistent cytokine production by synovial fibroblasts may play an important role in the continued recruitment and activation of inflammatory cells in chronic arthritis and in the formation of rheumatoid pannus.

Topics: Animals; Arthritis, Rheumatoid; Cell Division; Cell Line; Cells, Cultured; Cytokines; DNA Replication; Epidermal Growth Factor; Fibroblast Growth Factor 2; Fibroblasts; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Inflammation; Interleukin-1; Interleukin-6; Mice; Osteoarthritis; Synovial Membrane; Transforming Growth Factor beta

Monolayer culture of rabbit articular chondrocytes has been used to study the effects of transforming growth factor-beta (TGF-beta) and interleukin 1 (IL-1) on the production of matrix components, particularly collagens and proteoglycans. TGF-beta was shown to stimulate synthesis of collagen types II and XI as well as that of proteoglycans. The factor increases steady state level of procollagens I, II and III mRNA. Proteoglycans produced in the presence of TGF-beta had the same hydrodynamic sizes as those of controls, but a decrease of the ratio of chondroiting 6-sulfate: chondroitin 4-sulfate was observed. It was shown that TGF-beta may counteract the effect of IL-1 on synthesis of both collagen and proteoglycan and production of metalloproteases when it is introduced after IL-1, while it does not prevent the effect of the monokine when it is first added to the cultures.

Topics: Animals; Cartilage, Articular; Cells, Cultured; Collagen; Cytokines; Electrophoresis, Polyacrylamide Gel; Interleukin-1; Metalloendopeptidases; Osteoarthritis; Proteoglycans; Rabbits; Transforming Growth Factor beta

Topics: Aging; Animals; Animals, Newborn; Cartilage, Articular; Cytokines; Dogs; Glycosaminoglycans; Insulin-Like Growth Factor I; Interleukin-1; Kinetics; Osteoarthritis; Proteoglycans; Recombinant Proteins; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Rheumatoid arthritis (RA), and not osteoarthritis (OA) synovial cells proliferate in serum-free medium, a finding that suggests that, in vitro, RA synovial cells may be stimulated to grow by the continuous autocrine production of at least one polypeptide growth factor. Adding monoclonal antibody 1D11.16, or rabbit polyclonal anti-tumor growth factor beta (anti-TGF-beta) antibodies (both neutralizing antibodies to TGF-beta 1 and TGF-beta 2) to RA synovial cells, in culture, caused a significant reduction in cell growth, an effect not seen when other growth factor antibodies (platelet-derived growth factor [PDGF], epidermal growth factor [EGF], or EGF receptor) were added to the culture medium. Taken together, these data are consistent with the concept that RA synovial cell growth in vitro is driven endogenous TGF-beta. Moreover, when EGF was added to the culture medium, this caused the numbers of RA, and not OA, synovial cells to increase significantly. This finding suggests that RA synovial cells are in G1 phase of the cell cycle; an effect that could be mediated by endogenous TGF-beta.

Topics: Arthritis, Rheumatoid; Cell Division; Cell Line; Cell Survival; Humans; In Vitro Techniques; Osteoarthritis; Synovial Membrane; Transforming Growth Factor beta

When stimulated with increasing amounts of interleukin 1 beta (IL 1 beta) rheumatoid arthritis (RA), as compared with osteoarthritis (OA), synovial cells grown in RPMI plus fetal bovine serum (FBS), released significantly more prostaglandin E2 (PGE2) (p less than 0.05; paired t test, two-tailed). PGE2 release by IL 1 beta-stimulated RA synovial cells grown for 14 days in serum-free RPMI was significantly less than that released by the same cells grown in medium plus 10% FBS (p less than 0.03; two-tailed). Since these data suggest that growth factors present in FBS may augment the effects of IL 1 beta, experiments were conducted to study the influence of four polypeptide growth factors--transforming growth factor-beta (TGF-beta), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF), on IL 1 beta-induced release of PGE2 by cultured RA synovial cells. Both EGF and bFGF significantly enhanced IL 1 beta-induced release of PGE2 (p less than 0.05; paired t test, one-tailed), while PDGF was synergistic with IL 1 beta, significantly increasing release of PGE2 by these cultured cells (p less than 0.02; two-tailed). No such effect was seen when TGF-beta was added to the culture medium. Taken together, these data lend support to the concept that within the synovial micro-environment small quantities of individual growth factors may potentiate the effects of IL 1 beta to amplify intra-articular inflammation.

Topics: Arthritis, Rheumatoid; Cells, Cultured; Dinoprostone; Epidermal Growth Factor; Fibroblast Growth Factor 2; Growth Substances; Humans; In Vitro Techniques; Interleukin-1; Osteoarthritis; Platelet-Derived Growth Factor; Synovial Membrane; Transforming Growth Factor beta